Antisense IAP nucleobase oligomers and uses thereof

ABSTRACT

The present invention features nucleobase oligomers that hybridize to IAP polynucleotides, and methods for using them to enhance apoptosis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Application No.60/367,853, filed Mar. 27, 2002, which is hereby incorporated byreference.

BACKGROUND OF THE INVENTION

The invention relates to antisense IAP nucleobase oligomers and methodsof using them to induce apoptosis.

One way by which cells die is referred to as apoptosis, or programmedcell death. Apoptosis often occurs as a normal part of the developmentand maintenance of healthy tissues. The process may occur so rapidlythat it is difficult to detect.

The apoptosis pathway is now known to play a critical role in embryonicdevelopment, viral pathogenesis, cancer, autoimmune disorders, andneurodegenerative diseases, as well as other events. The failure of anapoptotic response has been implicated in the development of cancer,autoimmune disorders, such as lupus erythematosis and multiplesclerosis, and in viral infections, including those associated withherpes virus, poxvirus, and adenovirus.

The importance of apoptosis in cancer has become clear in recent years.The identification of growth promoting oncogenes in the late 1970's gaverise to an almost universal focus on cellular proliferation thatdominated research in cancer biology for many years. Long-standing dogmaheld that anti-cancer therapies preferentially targeted rapidly dividingcancer cells relative to “normal” cells. This explanation was notentirely satisfactory, since some slow growing tumors are easilytreated, while many rapidly dividing tumor types are extremely resistantto anti-cancer therapies. Progress in the cancer field has now led to anew paradigm in cancer biology wherein neoplasia is viewed as a failureto execute normal pathways of programmed cell death. Normal cellsreceive continuous feedback from their neighbors through various growthfactors, and commit “suicide” if removed from this context. Cancer cellssomehow bypass these commands and continue inappropriate proliferation.It is now believed that many cancer therapies, including radiation andmany chemotherapies, previously thought to act by causing cellularinjury, actually work by triggering apoptosis.

Both normal cell types and cancer cell types display a wide range ofsusceptibility to apoptotic triggers, although the determinants of thisresistance are only now under investigation. Many normal cell typesundergo temporary growth arrest in response to a sub-lethal dose ofradiation or cytotoxic chemical, while cancer cells in the vicinityundergo apoptosis. This differential effect at a given dose provides thecrucial treatment window that allows successful anti-cancer therapy. Itis therefore not surprising that resistance of tumor cells to apoptosisis emerging as a major category of cancer treatment failure.

Several potent endogenous proteins that inhibit apoptosis have beenidentified, including Bcl-2, and IAP (inhibitor-of apoptosis) familiesin mammalian cells. Certain members of the latter family directlyinhibit terminal effector caspases, i.e. casp-3 and casp-7, engaged inthe execution of cell death, as well as the key mitochondrial initiatorcaspase, casp-9, important to the mediation of cancer chemotherapyinduced cell death. The IAPs are the only known endogenous caspaseinhibitors, and thus play a central role in the regulation of apoptosis.

The IAPs have been postulated to contribute to the development of somecancers, and a postulated causal chromosomal translocation involving oneparticular IAP (cIAP2/HIAP1) has been identified in MALT lymphoma. Arecent correlation between elevated XIAP, poor prognosis, and shortsurvival has been demonstrated in patients with acute myelogenousleukemia. XIAP was highly over-expressed in many tumor cell lines of theNCI panel.

There exists a need for improved cancer therapeutics and, in particular,therapeutics that can induce cancer cells to undergo apoptosis andoverride anti-apoptotic signals provided in such cells.

SUMMARY OF THE INVENTION

In general, the invention features methods and reagents useful forinducing apoptosis in a cell. The methods and reagents of the inventionare useful in treating cancers, and other proliferative diseases.

The present invention features nucleobase oligomers, particularlyoligonucleotides, for use in modulating the function of a polynucleotideencoding an IAP. These oligomers reduce the amount of an IAP produced,allowing a cell normally expressing the IAP to undergo apoptosis. Thisis accomplished by providing nucleobase oligomers that specificallyhybridize with one or more polynucleotides encoding an IAP. The specifichybridization of the nucleobase oligomer with an IAP polynucleotide(e.g., RNA, DNA) interferes with the normal function of that IAPpolynucleotide, reducing the amount of IAP protein produced. Thismodulation of function of a target nucleic acid by compounds thatspecifically hybridize to the target is generally referred to as“antisense.”

In one aspect, the invention features a nucleobase oligomer of up to 30nucleobases in length, the oligomer including at least eight consecutivenucleobases of a sequence selected from SEQ ID NOs: 1-99, 143, 147, 151,163-260, 287, 289, and 300-460. Desirably, when administered to a cell,the oligomer inhibits expression of an IAP.

In certain embodiments, the nucleobase oligomer includes a sequenceselected from SEQ ID NOs: 1-99, 143, 147, 151, 163-260, 287, 289, and300-460. It is desirable that the nucleobase oligomer consists of (oressentially of) one or more of the foregoing SEQ ID NOs. For example,the nucleobase oligomer may be a XIAP antisense nucleic acid thatincludes a sequence chosen from SEQ ID NOs 97, 98, 99, 143, 147, 151,287, and 289, a HIAP1 antisense nucleic acid that includes a sequencechosen from SEQ ID NOs 300-389, or a HIAP2 antisense nucleic acidincludes a sequence chosen from SEQ ID NOs 390-460. In a particularlydesirable embodiment, the invention features a nucleobase oligomerhaving eleven DNA residues flanked on each side by four 2′-O-methyl RNAresidues, and consists of one of the following sequences: 5′-AUUGGTTCCAATGTGUUCU-3′ (SEQ ID NO: 155); 5′-ACACGACCGCTAAGAAACA-3′ (SEQ ID NO:16); 5′-ACAGGACTACCACTTGGAA-3′ (SEQ ID NO: 157); 5′-UGCCAGTGTTGATGCUGAA-3′ (SEQ ID NO: 27); 5′-GCUGAGTCTCCATATUGCC-3′ (SEQ ID NO:141); 5′-UCGGGTATATGGTGTCUGA-3′ (SEQ ID NO: 41); 5′-AAGCACTGCACTTGGUCAC-3′ (SEQ ID NO: 47); 5′- CCGGCCCAAAACAA AGAAG-3′ (SEQ ID NO:51); 5′- ACCCTGGATACCATTUAGC-3′ (SEQ ID NO: 63); 5′-UGUCAGTACATGTTGGCUC-3′ (SEQ ID NO: 161); and 5′-UGCACCCTGGATA CCAUUU-3′ (SEQ IDNO: 151).

A nucleobase oligomer of the present invention may include at least onemodified linkage (e.g., a phosphorothioate, a methylphosphonate, aphosphotriester, a phosphorodithioate, or a phosphoselenate linkage),modified nucleobase (e.g., a 5-methyl cytosine), and/or a modified sugarmoiety (e.g., a 2′-O-methoxyethyl group or a 2′-O-methyl group). In oneembodiment, the oligomer is a chimeric oligomer (e.g., anoligonucleotide that includes DNA residues linked together byphosphorothioate or phosphodiester linkages, flanked on each side by atleast one, two, three, or four 2′-O-methyl RNA residue linked togetherby a phosphorothioate linkage).

In another aspect, the invention features a method of enhancingapoptosis in a cell. This method includes the step of administering tothe cell a nucleobase oligomer of the present invention so thatexpression of an IAP (e.g., XIAP, HIAP1, or HIAP2) is inhibited. Thenucleobase oligomer may be, e.g., a component of an antisense compound,a double-stranded RNA, or a ribozyme. This administering step may beperformed alone, or in combination with a second step (e.g.,administration of a chemotherapeutic agent, a biological responsemodifying agent, and/or a chemosensitizer). The cell can be in vitro orin vivo. In one embodiment, the cell is a cancer cell (e.g., a humancancer cell) or a cell of lymphoid or myeloid origin.

In a related aspect, the invention features a method for treating ananimal (e.g., a human) having a proliferative disease (e.g., a cancer,lymphoproliferative disorder, or myelodysplastic syndrome) or preventingthe development of such a disease, by administering to the animal aneffective amount of a nucleobase oligomer of the present invention.

The cancer may be, for example, acute leukemia, acute lymphocyticleukemia, acute myelocytic leukemia, acute myeloblastic leukemia, acutepromyelocytic leukemia, acute myelomonocytic leukemia, acute monocyticleukemia, acute erythroleukemia, chronic leukemia, chronic myelocyticleukemia, myelodysplastic syndrome, chronic lymphocytic leukemia,polycythemia vera, lymphoma, Hodgkin's disease, Waldenstrom'smacroglobulinemia, fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinoma,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, uterinecancer, testicular cancer, lung carcinoma, small cell lung carcinoma,bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodenroglioma, schwannoma,meningioma, melanoma, neuroblastoma, or retinoblastoma. When treating acancer, it may be desirable to also administer one or morechemotherapeutic agents, biological response modifying agents, and/orchemosensitizers. Desirably, the administration of one or more of theseagents is within five days of the administration of the nucleobaseoligomer. Exemplary chemotherapeutic agents are adriamycin(doxorubicin), vinorelbine, etoposide, taxol, and cisplatin. While anyroute of administration that results in an effective amount at thedesired site may be used, particularly desirable routes are byintravenous and intratumoral administration.

In another aspect, the invention features a pharmaceutical compositionthat includes a nucleobase oligomer of the present invention and apharmaceutically acceptable carrier. If desirable, the pharmaceuticalcomposition may further include additional components (e.g., a.colloidal dispersion system or a chemotherapeutic agent).

The invention also features a catalytic RNA molecule capable of cleavingXIAP, HIAP1, or HIAP2 mRNA. In desirable embodiments, the catalytic RNAmolecule includes, in its binding arms, at least eight consecutivenucleobases corresponding to a nucleobase oligomer of the invention.(e.g., a nucleobase sequence of any one of Tables 1, 2, 6, and 7). TheRNA molecule is desirably in a hammerhead motif, but may also be in ahairpin, hepatitis delta virus, group 1 intron, VS RNA or RNAseP RNAmotif.

The invention also features an expression vector including a nucleicacid encoding one or more catalytic RNA molecules of the inventionpositioned for expression in a mammalian cell.

The invention also features a method of treating an animal having acancer or lymphoproliferative disorder by administering to the animal aneffective amount of a catalytic RNA molecule described above, or anexpression vector encoding such a catalytic RNA molecule.

In still another aspect, the invention features a double-stranded RNAmolecule having between 21 and 29 nucleobases, wherein at least eightconsecutive nucleobases corresponding to a sequence of any one of Tables1, 2, 6, and 7 are present.

In a related aspect, the invention also features a double-stranded RNAmolecule having between 50 and 70 nucleobases, the RNA molecule having afirst domain of between 21 and 29 nucleobases that include least eightconsecutive nucleobases corresponding to a sequence of any one of Tables1, 2, 6, and 7; a second domain complementary to the first domain, and aloop domain situated between the first and second domains such that thefirst and second domains are capable of duplexing to form adouble-stranded RNA molecule. The invention also features an expressionvector (e.g., an adenoviral vector or a retroviral vector) encoding sucha double stranded RNA.

The invention also features a method of treating an animal having acancer or lymphoproliferative disorder by administering to the animal aneffective amount of a double-stranded RNA molecule described above

By a “nucleobase oligomer” is meant a compound that includes a chain ofat least eight nucleobases joined together by linkage groups. Includedin this definition are natural and non-natural oligonucleotides, bothmodified and unmodified, as well as oligonucleotide mimetics such asProtein Nucleic Acids, locked nucleic acids, and arabinonucleic acids.Numerous nucleobases and linkage groups may be employed in thenucleobase oligomers of the invention, including those described indetail herein in the section entitled “Oligonucleotides and othernucleobase oligomers,” infra.

“Protein” or “polypeptide” or “polypeptide fragment” means any chain ofmore than two amino acids, regardless of post-translational modification(e.g., glycosylation or phosphorylation), constituting all or part of anaturally occurring polypeptide or peptide, or constituting anon-naturally occurring polypeptide or peptide.

“Apoptosis” means the process of cell death wherein a dying celldisplays a set of well-characterized biochemical hallmarks that includecell membrane blebbing, cell soma shrinkage, chromatin condensation, andDNA laddering. Cells that die by apoptosis include neurons (e.g., duringthe course of neurodegenerative diseases such as stroke, Parkinson'sdisease, and Alzheimer's disease), cardiomyocytes (e.g., aftermyocardial infarction or over the course of congestive heart failure),and cancer cells (e.g., after exposure to radiation or chemotherapeuticagents). Environmental stress (e.g., hypoxic stress) that is notalleviated may cause a cell to enter the early phase of the apoptoticpathway, which is reversible (i.e., cells at the early stage of theapoptotic pathway can be rescued). At a later phase of apoptosis (thecommitment phase), cells cannot be rescued, and, as a result, arecommitted to die.

Proteins and compounds known to stimulate and inhibit apoptosis in adiverse variety of cells are well known in the art. For example,intracellular expression and activation of the caspase (ICE) familyinduces or stimulates apoptotic cell death, whereas expression of theIAPs or some members of the Bcl-2 family inhibit apoptotic cell death.In addition, there are survival factors that inhibit cell death inspecific cell types. For example, neurotrophic factors, such as NGFinhibit neuronal apoptosis.

By “IAP gene” is meant a gene encoding a polypeptide having at least oneBIR domain and that is capable of modulating (inhibiting or enhancing)apoptosis in a cell or tissue when provided by other intracellular orextracellular delivery methods (see, e.g., U.S. Pat. No. 5,919,912). Inpreferred embodiments, the IAP gene is a gene having about 50% orgreater nucleotide sequence identity (e.g., at least 85%, 90%, or 95%)to at least one of human or murine XIAP, HIAP1, or HIAP2 (each of whichis described in U.S. Pat. No. 6,156,535). Preferably the region ofsequence over which identity is measured is a region encoding at leastone BIR domain and a ring zinc finger domain. Mammalian IAP genesinclude nucleotide sequences isolated from any mammalian source.Preferably the mammal is a human.

By “IAP protein” or “IAP polypeptide” is meant a polypeptide, orfragment thereof, encoded by an IAP gene.

By “IAP biological activity” is meant any activity known to be caused invivo or in vitro by an IAP polypeptide.

By “enhancing apoptosis” is meant increasing the number of cells thatapoptose in a given cell population (e.g., cancer cells, lymphocytes,fibroblasts, or any other cells). It will be appreciated that the degreeof apoptosis enhancement provided by an apoptosis-enhancing compound ina given assay will vary, but that one skilled in the art can determinethe statistically significant change in the level of apoptosis thatidentifies a nucleobase oligomer that enhances apoptosis otherwiselimited by an IAP. Preferably, “enhancing apoptosis” means that theincrease in the number of cells undergoing apoptosis is at least 10%,more preferably the increase is 25% or even 50%, and most preferably theincrease is at least one-fold, relative to cells not administered anucleobase oligomer of the invention but otherwise treated in asubstantially similar manner. Preferably the sample monitored is asample of cells that normally undergo insufficient apoptosis (i.e.,cancer cells). Methods for detecting changes in the level of apoptosis(i.e., enhancement or reduction) are described herein.

By a nucleobase oligomer that “inhibits the expression” of a target gene(e.g., an IAP) is meant one that reduces the amount of a target mRNA, orprotein encoded by such mRNA, by at least about 5%, more desirable by atleast about 10%, 25%, or even 50%, relative to an untreated control.Methods for measuring both mRNA and protein levels are well-known in theart; exemplary methods are described herein.

“Hybridization” means hydrogen bonding, which may be Watson-Crick,Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementarynucleobases. For example, adenine and thymine are complementarynucleobases that pair through the formation of hydrogen bonds.

By “proliferative disease” is meant a disease that is caused by orresults in inappropriately high levels of cell division, inappropriatelylow levels of apoptosis, or both. For example, cancer is an example of aproliferative disease. Examples of cancers include, without limitation,leukemias (e.g., acute leukemia, acute lymphocytic leukemia, acutemyelocytic leukemia, acute myeloblastic leukemia, acute promyelocyticleukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acuteerythroleukemia, chronic leukemia, chronic myelocytic leukemia, chroniclymphocytic leukemia), polycythemia vera, lymphoma (Hodgkin's disease,non-Hodgkin's disease), Waldenstrom's macroglobulinemia, heavy chaindisease, and solid tumors such as sarcomas and carcinomas (e.g.,fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, nile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, uterinecancer, testicular cancer, lung carcinoma, small cell lung carcinoma,bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodenroglioma, schwannoma,meningioma, melanoma, neuroblastoma, and retinoblastoma).Lymphoproliferative disorders are also considered to be proliferativediseases.

Preferably, a nucleobase oligomer of the invention is capable ofenhancing apoptosis and/or decreasing IAP mRNA or protein levels whenpresent in a cell that normally does not undergo sufficient apoptosis.Preferably the increase is by at least 10%, relative to a control, morepreferably 25%, and most preferably 1-fold or more. Preferably anucleobase oligomer of the invention includes from about 8 to 30nucleobases, wherein at least eight consecutive nucleobases are from asequence selected from SEQ ID NOs: 1-99, 143, 147, 151, 163-260, 287,289, and 300-460. A nucleobase oligomer of the invention may alsocontain, e.g., an additional 20, 40, 60, 85, 120, or more consecutivenucleobases that are complementary to an IAP polynucleotide. Thenucleobase oligomer (or a portion thereof) may contain a modifiedbackbone. Phosphorothioate, phosphorodithioate, and other modifiedbackbones are known in the art. The nucleobase oligomer may also containone or more non-natural linkages.

By “chemotherapeutic agent” is meant an agent that is used to killcancer cells or to slow their growth. Accordingly, both cytotoxic andcytostatic agents are considered to be chemotherapeutic agents.

By “biological response modifying agent” is meant an agent thatstimulates or restores the ability of the immune system to fightdisease. Some, but not all, biological response modifying agents mayslow the growth of cancer cells and thus are also considered to bechemotherapeutic agents.” Examples of biological response modifyingagents are interferons (alpha, beta, gamma), interleukin-2, rituximab,and trastuzumab.

By “chemosensitizer” is meant an agent that makes tumor cells moresensitive to the effects of chemotherapy.

By “an effective amount” is meant the amount of a compound (e.g., anucleobase oligomer) required to ameliorate the symptoms of a disease,inhibit the growth of the target cells, reduce the size or number oftumors, inhibit the expression of an IAP, or enhance apoptosis of targetcells, relative to an untreated patient. The effective amount of activecompound(s) used to practice the present invention for therapeutictreatment of abnormal proliferation (i.e., cancer) varies depending uponthe manner of administration, the age, body weight, and general healthof the subject. Ultimately, the attending physician or veterinarian willdecide the appropriate amount and dosage regimen. Such amount isreferred to as an “effective” amount.

By “lymphoproliferative disorder” is meant a disorder in which there isabnormal proliferation of cells of the lymphatic system (e.g., T-cellsand B-cells), and includes multiple sclerosis, Crohn's disease, lupuserythematosus, rheumatoid arthritis, and osteoarthritis.

By “ribozyme” is meant an RNA that has enzymatic activity, possessingsite specificity and cleavage capability for a target RNA molecule.Ribozymes can be used to decrease expression of a polypeptide. Methodsfor using ribozymes to decrease polypeptide expression are described,for example, by Turner et al., (Adv. Exp. Med. Biol. 465:303-318, 2000)and Norris et al., (Adv. Exp. Med. Biol. 465:293-301, 2000).

By “reporter gene” is meant a gene encoding a polypeptide whoseexpression may be assayed; such polypeptides include, withoutlimitation, glucuronidase (GUS), luciferase, chloramphenicoltransacetylase (CAT), and beta-galactosidase.

By “promoter” is meant a polynucleotide sufficient to directtranscription.

By “operably linked” is meant that a first polynucleotide is positionedadjacent to a second polynucleotide that directs transcription of thefirst polynucleotide when appropriate molecules (e.g., transcriptionalactivator proteins) are bound to the second polynucleotide.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1L are graphs showing the effect of antisense XIAPoligonucleotides on XIAP protein expression, relative to total protein(FIGS. 1A, 1C, 1E, 1G, 11, and 1K). FIGS. 1B, 1D, 1F, 1H, 1J, and 1L arethe total protein concentration values for each oligonucleotidetransfection compared to mock transfection results that were used tonormalize the above XIAP protein results.

FIGS. 2A-2C are graphs showing the effects of various antisense XIAPoligonucleotides, alone or in combination, on XIAP RNA (FIG. 2A) andprotein (FIG. 2B). FIG. 2C is a graph of the total protein concentrationvalues for each oligonucleotide transfection compared to mocktransfection results, which were used to normalize the XIAP proteinresults shown in FIG. 2B.

FIGS. 3 and 4 are graphs showing the effects of 4×4 mixed backbone (MBO)FG8 or E12 oligonucleotides in amounts of 31 nM (FIG. 3) or 63 nM (FIG.4). H460 lung carcinoma cells were transfected for 18 hours on one, two,or three consecutive days using 125 nM MBOs and Lipofectamine 2000.Samples for western analysis were harvested at the indicated time.Scanning densitometry was performed, and XIAP protein levels werenormalized to GAPDH and compared to a mock control set to 100%. Theindicated percentages express % XIAP protein knockdown versus specificscrambled controls.

FIGS. 5A-5D are graphs of the effects of antisense XIAP oligonucleotideson cell viability (FIGS. 5A, 5C, and 5D), and chemosensitization in thepresence of adriamycin (FIG. 5B).

FIG. 6 is a graph showing oligonucleotide-mediated specificdown-regulation of XIAP mRNA in H460 cells in vitro. Depicted are XIAPmRNA levels in H460 cells treated with Lipofectamine 2000 alone (LFA) orLipofectamine 2000 with 1.2 □M of oligonucleotides F3, G4, CS, AB6, DE4or D7, or a respective reverse polarity (RP) or scrambled (SC)oligonucleotide control. Real-time RT-PCR quantification of the relativeamount of XIAP mRNA was performed at 6 hours of transfection. All dataare presented as the mean±standard deviation (SD) of triplicates from arepresentative experiment. The level of XIAP mRNA in untreated cells(control) maintained under identical experimental conditions wasassigned a value of 1.

FIG. 7 is a graph showing XIAP RNA levels in H460 cells aftertransfection with various PS-XIAP oligonucleotides. H460 human lungcancer cells were transfected for 6 hours using 1 μM PS-oligonucleotidesand Lipofectamine 2000. Cells were then harvested for Taqman analysis.

FIG. 8 is a graph showing XIAP RNA levels in H460 cells 9 hourspost-transfection with 4×4 MBOs. H460 cells were transfected for 9 hoursusing 4×4 MBOs at 62.5 nM to 1 μM and Lipofectamine 2000. The cells werethen harvested for Taqman analysis.

FIG. 9 is a graph showing XIAP protein knockdown in H460 cells 24 hoursafter transfection with 4×4 MBOs. H460 cells were transfected for 24hours using 1 μM 4×4 MBOs at 1 μM and Lipofectamine 2000. The cells werethen harvested for western blot analysis. Scanning densitometry wasperformed, and XIAP protein levels were normalized to actin and comparedto their specific scrambled (sm, rm) controls, which were set at 100%.

FIGS. 10A and 10B are schematic illustrations showing antisense-mediatedspecific downregulation of XIAP protein in H460 cells in vitro. Depictedare XIAP protein levels in H460 cells treated with Lipofectamine 2000alone (LFA) or LFA plus 1.2 μM of XIAP oligonucleotides F3, G4, or C5,or their respective oligonucleotide controls (RP, SC). XIAP proteinlevels were analyzed by western blotting (FIG. I OA), and the amount ofprotein was quantified by densitometry (FIG. 10B). XIAP levels werenormalized to cellular actin levels and compared to untreated control(CNT) levels.

FIGS. 11A and 11B are schematic illustrations showing XIAPoligonucleotide-mediated effects on caspase activation. The effect ofXIAP oligonucleotides F3, G4, or CS, or their respective RP and SC ODNcontrols at 1.2 μM on the expression of pro-caspase-3, PARP (both fulllength (116 kDa) and processed (85 kDa)) (FIG. 10A) and Bcl-2 and Baxprotein levels (FIG. 10B) in transfected H460 cells compared to controlis shown. Proteins expression was analyzed by western blotting. Bcl-2and Bax protein levels were normalized to cellular actin levels andquantified by densitometry. The ratio of Bcl-2/Bax is presented as themean of two or three independent experiments, and the ratio in control(CNT) cells set at 1.

FIGS. 12A and 12B are schematic illustrations showing XIAPoligonucleotide-specific induction of apoptosis. Induction of apoptosiswas measured in H460 cells treated with 1.2 μM of XIAP G4 ASoligonucleotide, G4 SC oligonucleotide or untreated control (CNT). FIG.12A shows the percentage of cells having sub-G0/G1 (apoptotic) DNAcontent, as measured by propidium iodide (PI) staining and flowcytometry. FIG. 12B. shows nuclear morphology of oligonucleotide-treatedH460 cells stained with DAPI. Arrows indicate cells that havecharacteristic apoptotic morphology of nuclear DNA condensation orfragmentation.

FIG. 13A is a graph showing the effect of XIAP G4 AS oligonucleotidetreatment on the viability of H460 cells. Cells were treated with anincreasing concentration of LFA alone or LFA-oligonucleotide complexeswith G4 AS oligonucleotides or G4 SC oligonucleotides, and cellsviability was determined by MTT assay after 24 hours of treatment. Thedata represent the mean±SD of three independent experiments.

FIG. 13B is a graph showing the percentage of dead H460 cells aftertreatment with LFA and complexes with G4 AS oligonucleotides or G4 SColigonucleotides at 0.4 μM dose in the presence or absence ofdoxorubicin (DOX), taxol, vinorelbine (VNB) or etoposide (Etop), asdetermined by MTT assay. The data represent the mean±SD of threeindependent experiments.

FIG. 14 is a graph showing relative H460 tumor growth in mice treatedwith XIAP AS 2×2 MBOs and vinorelbine. Intratumoral injection ofoligonucleotides at 50 μg/g tumor mass was performed in SCID-RAG2 micecarrying subcutaneous H460 cell xenografts. This treatment was combinedwith administration of vinorelbine.

FIG. 15 is a graph showing mean H460 cell tumor size in mice treatedsystemically with XIAP AS PS-oligonucleotides. Systemic delivery (i.p.)of XIAP AS PS-oligonucleotides into SCID-RAG2 mice implanted withsubcutaneous H460 cell xenografts reduced the size of the tumors,relative to control.

FIG. 16 is a graph showing MDA-MB-435/LCC6 human breast carcinoma cell(LCC cell) tumor size in mice treated systemically with XIAP ASPS-oligonucleotides. Systemic delivery (i.p.) of XIAP ASPS-oligonucleotides into female SCID-RAG2 mice implanted with LCC6 cellxenografts in mammary fat pads reduced the size of the tumors, relativeto control.

FIG. 17 is a schematic illustration showing in vivo effects of G4oligonucleotides on tumor growth and tumor XIAP protein levels.Antitumor efficacy of systemically delivered, naked XIAP G4 ASoligonucleotides or G4 SC oligonucleotides on the growth of subcutaneousH460 cell xenografts in male SCID-RAG2 mice. All data are expressed asmean±SEM (n=6 mice/group).

FIGS. 18A and 18B are schematic illustrations depicting XIAP proteinexpression levels in H460 tumor xenografts implanted in SCID-RAG2 miceafter 21 days treatment with G4 AS oligonucleotides, G4 SColigonucleotides, or vehicle alone (control), analyzed by westernblotting and quantified by densitometry. XIAP levels were normalized tocellular actin levels. All data are expressed as mean±SD (n=3).

FIGS. 19A and 19B are photomicrographs showing in vivo effects of G4oligonucleotides on histopathology of H460 tumors implanted in SCID-RAG2mice after 15 mg/kg systemic dosing of XIAP G4 AS oligonucleotides or G4SC oligonucleotides over 21 days. FIG. 19A depicts tumor sectionsstained with hematoxylin and eosin. FIG. 19B shows immunohistochemistryof ubiquitin expression in tumor sections. Representative tumorphotomicrographs are shown. Internal scale markers equal 100 μm.

FIGS. 20A and 20B are graphs showing increased in vivo efficacy ofvinorelbine (VNB) in combination with XIAP oligonucleotides. Antitumorefficacy of VNB with or without XIAP G4 AS oligonucleotides or G4 SColigonucleotides against H460 tumors xenografts was determined inSCID-RAG2 mice. FIG. 20A depicts antitumor activity of single agents,while FIG. 20B depicts antitumor activity of VNB and G4 oligonucleotidesin combination. All data are expressed as means±SEM (n=6 mice/group).

FIG. 21 is a graph showing the effects of HIAP1 oligonucleotides onHIAP1 RNA levels.

FIGS., 22 A and 22B are schematic illustrations showing densitometricscans of western blots showing the effects of HIAP1 oligonucleotides ona cell's ability to block cycloheximide-induced upregulation of HIAP1protein.

FIG. 23 is a graph showing the effects of HIAP1 oligonucleotides oncytotoxicity, as measured by total protein.

FIG. 24 is a graph showing the validation of the sequence specificityfor HIAP1 oligonucleotide APO 2.

FIG. 25 is a graph showing the effect of HIAP1 oligonucleotides on thechemosensitization of drug-resistant SF295 glioblastomas.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides nucleobase oligomers that inhibitexpression of an LAP, and methods for using them to induce apoptosis ina cell. The nucleobase oligomers of the present invention may also beused to form pharmaceutical compositions. The invention also featuresmethods for enhancing apoptosis in a cell by administering anoligonucleotide of the invention in combination with a chemotherapeuticagent such as a cytotoxic agent, cytostatic agent, or biologicalresponse modifying agent (e.g., adriamycin, vinorelbine, etoposide,taxol, cisplatin, interferon, interleukin-2, monoclonal antibodies). Thechemotherapeutic agent may be, for example,. If desirable, achemosensitizer (i.e., an agent that makes the proliferating cells moresensitive to the chemotherapy) may also be administered. Any combinationof the foregoing agents may also be used to form a pharmaceuticalcomposition. These pharmaceutical compositions may be used to treat aproliferative disease, for example, cancer or a lymphoproliferativedisorder, or a symptom of a proliferative disease. The nucleobaseoligomer of the invention may also be used in combination withradiotherapy for the treatment of cancer or other proliferative disease.

Activation of apoptosis in cancer cells offers novel, and potentiallyuseful approaches to improve patient responses to conventionalchemotherapy or radiotherapy. XIAP is the most potent member of the IAPgene family in terms of its ability to directly inhibit caspases and tosuppress apoptosis. We investigated the effect of XIAP down-regulationby antisense (AS) oligonucleotides on human non-small cell lung cancer(NIH-H460) growth in vitro and in vivo. In cultured H460 human lungcancer cells, oligonucleotide G4 AS was identified as the most potentcompound, effectively down-regulated XIAP mRNA by 55% and protein levelsup to 60%, as determined by real-time RT-PCR and western blotting,respectively, and induced 60% cell death at 1.2 μM concentrations. Incontrast, the scrambled control G4 oligonucleotide caused little XIAPloss and less than 10% cell death. Treatment with G4 AS inducedapoptosis, as revealed by degradation of pro-caspase-3 and PARPproteins, with significant nuclear DNA condensation and fragmentation at1.2 μM concentrations. Moreover, XIAP AS oligonucleotides sensitizedH460 cells to the cytotoxic effects of doxorubicin, taxol, vinorelbine,and etoposide. In animal models, we demonstrate that G4 AS at 15 mg/kghad significant sequence-specific growth inhibitory effects on humanH460 tumors in xenograft models of SCID/RAG2-immunodeficient mice bysystemic intraperitoneal administration. Systemic AS ODN administrationwas associated with an 85% down-regulation of XIAP protein in tumorxenografts. The combination of 15 mg/kg G4 AS with 5 mg/kg vinorelbinesignificantly inhibited tumor growth, more than either agent alone.These studies indicate that down-regulation of XIAP is a potent deathsignal in lung carcinoma cells and is able to induce apoptosis in vitroas well as inhibit tumor growth in vivo. These studies support thecontention that lAPs are suitable targets for cancer therapy in humannon-small cell lung cancer, as well as other solid tumors.

Therapy

Therapy may be provided wherever cancer therapy is performed: at home,the doctor's office, a clinic, a hospital's outpatient department, or ahospital. Treatment generally begins at a hospital so that the doctorcan observe the therapy's effects closely and make any adjustments thatare needed. The duration of the therapy depends on the kind of cancerbeing treated, the age and condition of the patient, the stage and typeof the patient's disease, and how the patient's body responds to thetreatment. Drug administration may be performed at different intervals(e.g., daily, weekly, or monthly). Therapy may be given in on-and-offcycles that include rest periods so that the patient's body has a chanceto build healthy new cells and regain its strength.

Depending on the type of cancer and its stage of development, thetherapy can be used to slow the spreading of the cancer, to slow thecancer's growth, to kill or arrest cancer cells that may have spread toother parts of the body from the original tumor, to relieve symptomscaused by the cancer, or to prevent cancer in the first place.

As used herein, the terms “cancer” or “neoplasm” or “neoplastic cells”is meant a collection of cells multiplying in an abnormal manner. Cancergrowth is uncontrolled and progressive, and occurs under conditions thatwould not elicit, or would cause cessation of, multiplication of normalcells.

A nucleobase oligomer of the invention, or other negative regulator ofthe IAP anti-apoptotic pathway, may be administered within apharmaceutically-acceptable diluent, carrier, or excipient, in unitdosage form. Conventional pharmaceutical practice may be employed toprovide suitable formulations or compositions to administer thecompounds to patients suffering from a disease that is caused byexcessive cell proliferation. Administration may begin before thepatient is symptomatic. Any appropriate route of administration may beemployed, for example, administration may be parenteral, intravenous,intraarterial, subcutaneous, intratumoral, intramuscular, intracranial,intraorbital, ophthalmic, intraventricular, intrahepatic, intracapsular,intrathecal, intracisternal, intraperitoneal, intranasal, aerosol,suppository, or oral administration. For example, therapeuticformulations may be in the form of liquid solutions or suspensions; fororal administration, formulations may be in the form of tablets orcapsules; and for intranasal formulations, in the form of powders, nasaldrops, or aerosols.

Methods well known in the art for making formulations are found, forexample, in “Remington: The Science and Practice of Pharmacy” Ed. A. R.Gennaro, Lippincourt Williams & Wilkins, Philadelphia, Pa., 2000.Formulations for parenteral administration may, for example, containexcipients, sterile water, or saline, polyalkylene glycols such aspolyethylene glycol, oils of vegetable origin, or hydrogenatednapthalenes. Biocompatible, biodegradable lactide polymer,lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylenecopolymers may be used to control the release of the compounds. Otherpotentially useful parenteral delivery systems for IAP modulatorycompounds include ethylene-vinyl acetate copolymer particles, osmoticpumps, implantable infusion systems, and liposomes. Formulations forinhalation may contain excipients, for example, lactose, or may beaqueous solutions containing, for example, polyoxyethylene-9-laurylether, glycocholate and deoxycholate, or may be oily solutions foradministration in the form of nasal drops, or as a gel.

The formulations can be administered to human patients intherapeutically effective amounts (e.g., amounts which prevent,eliminate, or reduce a pathological condition) to provide therapy for adisease or condition. The preferred dosage of a nucleobase oligomer ofthe invention is likely to depend on such variables as the type andextent of the disorder, the overall health status of the particularpatient, the formulation of the compound excipients, and its route ofadministration.

As described above, if desired, treatment with a nucleobase oligomer ofthe invention may be combined with therapies for the treatment ofproliferative disease (e.g., radiotherapy, surgery, or chemotherapy).

For any of the methods of application described above, a nucleobaseoligomer of the invention is desirably administered intravenously or isapplied to the site of the needed apoptosis event (e.g., by injection).

Oligonucleotides and Other Nucleobase Oligomers

At least two types of oligonucleotides induce the cleavage of RNA byRNase H: polydeoxynucleotides with phosphodiester (PO) orphosphorothioate (PS) linkages. Although 2′-OMe-RNA sequences exhibit ahigh affinity for RNA targets, these sequences are not substrates forRNase H. A desirable oligonucleotide is one based on 2′-modifiedoligonucleotides containing oligodeoxynucleotide gaps with some or allinternucleotide linkages modified to phosphorothioates for nucleaseresistance. The presence of methylphosphonate modifications increasesthe affinity of the oligonucleotide for its target RNA and thus reducesthe IC₅₀. This modification also increases the nuclease resistance ofthe modified oligonucleotide. It is understood that the methods andreagents of the present invention may be used in conjunction with anytechnologies that may be developed, including covalently-closed multipleantisense (CMAS) oligonucleotides (Moon et al., Biochem J. 346:295-303,2000; PCT Publication No. WO 00/61595), ribbon-type antisense (RiAS)oligonucleotides (Moon et al., J. Biol. Chem. 275:4647-4653, 2000; PCTPublication No. WO 00/61595), and large circular antisenseoligonucleotides (U.S. Patent Application Publication No. U.S.2002/0168631 A1).

As is known in the art, a nucleoside is a nucleobase-sugar combination.The base portion of the nucleoside is normally a heterocyclic base. Thetwo most common classes of such heterocyclic bases are the purines andthe pyrimidines. Nucleotides are nucleosides that further include aphosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxylmoiety of the sugar. In forming oligonucleotides, the phosphate groupscovalently link adjacent nucleosides to one another to form a linearpolymeric compound. In turn, the respective ends of this linearpolymeric structure can be further joined to form a circular structure;open linear structures are generally preferred. Within theoligonucleotide structure, the phosphate groups are commonly referred toas forming the backbone of the oligonucleotide. The normal linkage orbackbone of RNA and DNA is a 3′ to 5′ phosphodiester linkage.

Specific examples of preferred nucleobase oligomers useful in thisinvention include oligonucleotides containing modified backbones ornon-natural internucleoside linkages. As defined in this specification,nucleobase oligomers having modified backbones include those that retaina phosphorus atom in the backbone and those that do not have aphosphorus atom in the backbone. For the purposes of this specification,modified oligonucleotides that do not have a phosphorus atom in theirinternucleoside backbone are also considered to be nucleobase oligomers.

Nucleobase oligomers that have modified oligonucleotide backbonesinclude, for example, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters,methyl and other alkyl phosphonates including 3′-alkylene phosphonatesand chiral phosphonates, phosphinates, phosphoramidates including3′-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′linkages, 2′-5′ linked analogs of these, and those having invertedpolarity, wherein the adjacent pairs of nucleoside units are linked3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and freeacid forms are also included. Representative United States patents thatteach the preparation of the above phosphorus-containing linkagesinclude, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863;4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019;.5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496;5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306;5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050, each of whichis herein incorporated by reference.

Nucleobase oligomers having modified oligonucleotide backbones that donot include a phosphorus atom therein have backbones that are formed byshort chain alkyl or cycloalkyl internucleoside linkages, mixedheteroatom and alkyl or cycloalkyl internucleoside linkages, or one ormore short chain heteroatomic or heterocyclic internucleoside linkages.These include those having morpholino linkages (formed in part from thesugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxideand sulfone backbones; formacetyl and thioformacetyl backbones;methylene formacetyl and thioformacetyl backbones; alkene containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, S and CH₂ component parts. RepresentativeUnited States patents that teach the preparation of the aboveoligonucleotides include, but are not limited to, U.S. Pat. Nos.:5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; and 5,677,439, each of which is hereinincorporated by reference.

In other nucleobase oligomers, both the sugar and the internucleosidelinkage, i.e., the backbone, are replaced with novel groups. Thenucleobase units are maintained for hybridization with an IAP. One suchnucleobase oligomer, is referred to as a Peptide Nucleic Acid (PNA). InPNA compounds, the sugar-backbone of an oligonucleotide is replaced withan amide containing backbone, in particular an aminoethylglycinebackbone. The nucleobases are retained and are bound directly orindirectly to aza nitrogen atoms of the amide portion of the backbone.Methods for making and using these nucleobase oligomers are described,for example, in “Peptide Nucleic Acids: Protocols and Applications” Ed.P. E. Nielsen, Horizon Press, Norfolk, United Kingdom, 1999.Representative United States patents that teach the preparation of PNAsinclude, but are not limited to, U.S. Pat. Nos.: 5,539,082; 5,714,33 1;and 5,719,262, each of which is herein incorporated by reference.Further teaching of PNA compounds can be found in Nielsen et al.,Science, 1991, 254, 1497-1500.

In particular embodiments of the invention, the nucleobase oligomershave phosphorothioate backbones and nucleosides with heteroatombackbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— (knownas a methylene (methylimino) or MMI backbone), —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂—, and —O—N(CH₃)—CH₂—CH₂—. In other embodiments,the oligonucleotides have morpholino backbone structures described inU.S. Pat. No. 5,034,506.

Nucleobase oligomers may also contain one or more substituted sugarmoieties. Nucleobase oligomers comprise one of the following at the 2′position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- orN-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl, and alkynylmay be substituted or unsubstituted C₁ to C₁₀ alkyl or —C₂ to C₁₀alkenyl and alkynyl. Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃,O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10.Other preferred nucleobase oligomers include one of the following at the2′ position: C, to C₁ to C₁₀ lower alkyl, substituted lower alkyl,alkaryl, aralkyl, O-alkaryl, or 0-aralkyl, SH, SCH₃, OCN, Cl, Br, CN,CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, NH₂, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl,an RNA cleaving group, a reporter group, an intercalator, a group forimproving the pharmacokinetic properties of a nucleobase oligomer, or agroup for improving the pharmacodynamic properties of an nucleobaseoligomer, and other substituents having similar properties. Preferredmodifications are 2′-O-methyl and 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃,also known as 2′-O-(2-methoxyethyl) or 2′-MOE). Another desirablemodification is 2′-dimethylaminooxyethoxy (i.e., O(CH₂)₂ON(CH₃)₂), alsoknown as 2′-DMAOE. Other modifications include, 2′-aminopropoxy(2′—OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similar modifications may alsobe made at other positions on an oligonucleotide or other nucleobaseoligomer, particularly the 3′ position of the sugar on the 3′ terminalnucleotide or in 2′-5′ linked oligonucleotides and the 5′ position of 5′terminal nucleotide. Nucleobase oligomers may also have sugar mimeticssuch as cyclobutyl moieties in place of the pentofuranosyl sugar.Representative United States patents that teach the preparation of suchmodified sugar structures include, but are not limited to, U.S. Pat.Nos.: 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137;5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722;5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873;5,670,633; and 5,700,920, each of which is herein incorporated byreference in its entirety.

Nucleobase oligomers may also include nucleobase modifications orsubstitutions. As used herein, “unmodified” or “natural” nucleobasesinclude the purine bases adenine (A) and guanine (G), and the pyrimidinebases thymine (T), cytosine (C) and uracil (U). Modified nucleobasesinclude other synthetic and natural nucleobases, such as5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine,hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives ofadenine and guanine; 2-propyl and other alkyl derivatives of adenine andguanine; 2-thiouracil, 2-thiothymine and 2-thiocytosine; 5-halouraciland cytosine; 5-propynyl uracil and cytosine; 6-azo uracil, cytosine andthymine; 5-uracil (pseudouracil); 4-thiouracil; 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines andguanines; 5-halo (e.g., 5-bromo), 5-trifluoromethyl and other5-substituted uracils and cytosines; 7-methylguanine and7-methyladenine; 8-azaguanine and 8-azaadenine; 7-deazaguanine and7-deazaadenine; and 3-deazaguanine and 3-deazaadenine. Furthernucleobases include those disclosed in U.S. Pat. No. 3,687,808, thosedisclosed in The Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons,1990, those disclosed by Englisch et al., Angewandte Chemie,International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y.S., Chapter 15, Antisense Research and Applications, pages 289-302,Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993. Certain of thesenucleobases are particularly usefil for increasing the binding affinityof an antisense oligonucleotide of the invention. These include5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6 and O-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2.degree. C.(Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds., Antisense Researchand Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and aredesirable base substitutions, even more particularly when combined with2′-O-methoxyethyl or 2′-O-methyl sugar modifications. RepresentativeUnited States patents that teach the preparation of certain of the abovenoted modified nucleobases as well as other modified nucleobases includeU.S. Pat. Nos.: 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066;5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711;5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941; and5,750,692, each of which is herein incorporated by reference.

Another modification of a nucleobase oligomer of the invention involveschemically linking to the nucleobase oligomer one or more moieties orconjugates that enhance the activity, cellular distribution, or cellularuptake of the oligonucleotide. Such moieties include but are not limitedto lipid moieties such as a cholesterol moiety (Letsinger et al., Proc.Natl. Acad. Sci. USA, 86:6553-6556, 1989), cholic acid (Manoharan etal., Bioorg. Med. Chem. Let, 4:1053-1060, 1994), a thioether, e.g.,hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci.,660:306-309, 1992; Manoharan et al., Bioorg. Med. Chem. Let.,3:2765-2770, 1993), a thiocholesterol (Oberhauser et al., Nucl. AcidsRes., 20:533-538: 1992), an aliphatic chain, e.g., dodecandiol orundecyl residues (Saison-Behmoaras et al., EMBO J., 10:1111-1118, 1991;Kabanov et al., FEBS Lett., 259:327-330, 1990; Svinarchuk et al.,Biochimie, 75:49-54, 1993), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 36:3651-3654, 1995; Shea et al., Nucl. Acids Res.,18:3777-3783, 1990), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 14:969-973, 1995), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett.,36:3651-3654, 1995), a palmityl moiety (Mishra et al., Biochim. Biophys.Acta, 1264:229-237, 1995), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 277:923-937, 1996. Representative United States patents thatteach the preparation of such nucleobase oligomer conjugates includeU.S. Pat. Nos.: 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,828,979; 4,835,263; 4,876,335; 4,904,582; 4,948,882;4,958,013; 5,082,830; 5,109,124; 5,112,963; 5,118,802; 5,138,045;5,214,136; 5,218,105; 5,245,022; 5,254,469; 5,258,506; 5,262,536;5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,414,077;5,416,203, 5,451,463; 5,486,603; 5,510,475; 5,512,439; 5,512,667;5,514,785; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,565,552;5,567,810; 5,574,142; 5,578,717; 5,578,718; 5,580,731; 5,585,481;5,587,371; 5,591,584; 5,595,726; 5,597,696; 5,599,923; 5,599,928;5,608,046; and 5,688,941, each of which is herein incorporated byreference.

The present invention also includes nucleobase oligomers that arechimeric compounds. “Chimeric” nucleobase oligomers are nucleobaseoligomers, particularly oligonucleotides, that contain two or morechemically distinct regions, each made up of at least one monomer unit,i.e., a nucleotide in the case of an oligonucleotide. These nucleobaseoligomers typically contain at least one region where the nucleobaseoligomer is modified to confer, upon the nucleobase oligomer, increasedresistance to nuclease degradation, increased cellular uptake, and/orincreased binding affinity for the target nucleic acid. An additionalregion of the nucleobase oligomer may serve as a substrate for enzymescapable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNaseH is a cellular endonuclease which cleaves the RNA strand of an RNA:DNAduplex. Activation of RNase H, therefore, results in cleavage of the RNAtarget, thereby greatly enhancing the efficiency of nucleobase oligomerinhibition of gene expression. Consequently, comparable results canoften be obtained with shorter nucleobase oligomers when chimericnucleobase oligomers are used, compared to phosphorothioatedeoxyoligonucleotides hybridizing to the same target region.

Chimeric nucleobase oligomers of the invention may be formed ascomposite structures of two or more nucleobase oligomers as describedabove. Such nucleobase oligomers, when oligonucleotides, have also beenreferred to in the art as hybrids or gapmers. Representative UnitedStates patents that teach the preparation of such hybrid structuresinclude U.S. Pat. Nos.: 5,013,830; 5,149,797; 5,220,007; 5,256,775;5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355;5,652,356; and 5,700,922, each of which is herein incorporated byreference in its entirety.

The nucleobase oligomers used in accordance with this invention may beconveniently and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by severalvendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is well known to usesimilar techniques to prepare oligonucleotides such as thephosphorothioates and alkylated derivatives.

The nucleobase oligomers of the invention may also be admixed,encapsulated, conjugated or otherwise associated with other molecules,molecule structures or mixtures of compounds, as for example, liposomes,receptor targeted molecules, oral, rectal, topical or otherformulations, for assisting in uptake, distribution and/or absorption.Representative United States patents that teach the preparation of suchuptake, distribution and/or absorption assisting formulations includeU.S. Pat. Nos.: 5,108,921; 5,354,844; 5,416,016; 5,459,127; 5,521,291;5,543,158; 5,547,932; 5,583,020; 5,591,721; 4,426,330; 4,534,899;5,013,556; 5,108,921; 5,213,804; 5,227,170; 5,264,221; 5,356,633;5,395,619; 5,416,016; 5,417,978; 5,462,854; 5,469,854; 5,512,295;5,527,528; 5,534,259; 5,543,152; 5,556,948; 5,580,575; and 5,595,756,each of which is herein incorporated by reference.

The nucleobase oligomers of the invention encompass any pharmaceuticallyacceptable salts, esters, or salts of such esters, or any other compoundthat, upon administration to an animal, is capable of providing(directly or indirectly) the biologically active metabolite or residuethereof. Accordingly, for example, the disclosure is also drawn toprodrugs and pharmaceutically acceptable salts of the compounds of theinvention, pharmaceutically acceptable salts of such prodrugs, and otherbioequivalents.

The term “prodrug” indicates a therapeutic agent that is prepared in aninactive form that is converted to an active form (i.e., drug) withinthe body or cells thereof by the action of endogenous enzymes or otherchemicals and/or conditions. In particular, prodrug versions of theoligonucleotides of the invention can be prepared as SATE[(S-acetyl-2-thioethyl) phosphate] derivatives according to the methodsdisclosed in PCT publication Nos. WO 93/24510 or WO 94/26764.

The term “pharmaceutically acceptable salts” refers to salts that retainthe desired biological activity of the parent compound and do not impartundesired toxicological effects thereto. Pharmaceutically acceptablebase addition salts are formed with metals or amines, such as alkali andalkaline earth metals or organic amines. Examples of metals used ascations are sodium, potassium, magnesium, calcium, and the like.Examples of suitable amines are N,N′-dibenzylethylenediamine,chloroprocaine, choline, diethanolamine, dicyclohexylamine,ethylenediamine, N-methylglucamine, and procaine (see, for example,Berge et al., J. Pharma Sci., 66:1-19, 1977). The base addition salts ofacidic compounds are prepared by contacting the free acid form with asufficient amount of the desired base to produce the salt in theconventional manner. The free acid form may be regenerated by contactingthe salt form with an acid and isolating the free acid in theconventional manner. The free acid forms differ from their respectivesalt forms somewhat in certain physical properties such as solubility inpolar solvents, but otherwise the salts are equivalent to theirrespective free acid for purposes of the present invention. As usedherein, a “pharmaceutical addition salt” includes a pharmaceuticallyacceptable salt of an acid form of one of the components of thecompositions of the invention. These include organic or inorganic acidsalts of the amines. Preferred acid salts are the hydrochlorides,acetates, salicylates, nitrates and phosphates. Other suitablepharmaceutically acceptable salts are well known to those skilled in theart and include basic salts of a variety of inorganic and organic acids,such as, for example, with inorganic acids, such as for examplehydrochloric acid, hydrobromic acid, sulfuric acid or phosphoric acid;with organic carboxylic, sulfonic, sulfo or phospho acids orN-substituted sulfamic acids, for example acetic acid, propionic acid,glycolic acid, succinic acid, maleic acid, hydroxymaleic acid,methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid,oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, salicylic acid,4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid,embonic acid, nicotinic acid or isonicotinic acid; and with amino acids,such as the 20 alpha-amino acids involved in the synthesis of proteinsin nature, for example glutamic acid or aspartic acid, and also withphenylacetic acid, methanesulfonic acid, ethanesulfonic acid,2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid,benzenesulfonic acid, 4-methylbenzenesulfonic acid,naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2- or3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid (withthe formation of cyclamates), or with other acid organic compounds, suchas ascorbic acid. Pharmaceutically acceptable salts of compounds mayalso be prepared with a pharmaceutically acceptable cation. Suitablepharmaceutically acceptable cations are well known to those skilled inthe art and include alkaline, alkaline earth, ammonium and quaternaryammonium cations. Carbonates or hydrogen carbonates are also possible.

For oligonucleotides and other nucleobase oligomers, suitablepharmaceutically acceptable salts include (i) salts formed with cationssuch as sodium, potassium, ammonium, magnesium, calcium, polyamines suchas spermine and spermidine, etc.; (ii) acid addition salts formed withinorganic acids, for example hydrochloric acid, hydrobromic acid,sulfuric acid, phosphoric acid, nitric acid and the like; (iii) saltsformed with organic acids such as, for example, acetic acid, oxalicacid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconicacid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid,palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonicacid, methanesulfonic acid, p-toluenesulfonic acid,naphthalenedisulfonic acid, polygalacturonic acid, and the like; and(iv) salts formed from elemental anions such as chlorine, bromine, andiodine.

The present invention also includes pharmaceutical compositions andformulations that include the nucleobase oligomers of the invention. Thepharmaceutical compositions of the present invention may be administeredin a number of ways depending upon whether local or systemic treatmentis desired and upon the area to be treated. Administration may betopical (including ophthalmic and to mucous membranes including vaginaland rectal delivery), pulmonary, e.g., by inhalation or insufflation ofpowders or aerosols, including by nebulizer; intratracheal, intranasal,epidermal and transdermal), oral, or parenteral. Parenteraladministration includes intravenous, intraarterial, subcutaneous,intraperitoneal, or intramuscular injection or infusion; orintracranial, e.g., intrathecal or intraventricular, administration.

Locked Nucleic Acids

Locked nucleic acids (LNAs) are nucleobase oligomers that can beemployed in the present invention. LNAs contain a 2′O, 4′-C methylenebridge that restrict the flexibility of the ribofuranose ring of thenucleotide analog and locks it into the rigid bicyclic N-typeconformation. LNAs show improved resistance to certain exo- andendonucleases and activate RNAse H, and can be incorporated into almostany nucleobase oligomer. Moreover, LNA-containing nucleobase oligomerscan be prepared using standard phosphoramidite synthesis protocols.Additional details regarding LNAs can be found in PCT publication No. WO99/14226 and U.S. Patent Application Publication No. U.S. 2002/0094555A1, each of which is hereby incorporated by reference.

Arabinonucleic Acids

Arabinonucleic acids (ANAs) can also be employed in methods and reagentsof the present invention. ANAs are nucleobase oligomers based onD-arabinose sugars instead of the natural D-2′-deoxyribose sugars.Underivatized ANA analogs have similar binding affinity for RNA as dophosphorothioates. When the arabinose sugar is derivatized with fluorine(2° F-ANA), an enhancement in binding affinity results, and selectivehydrolysis of bound RNA occurs efficiently in the resulting ANA/RNA andF-ANA/RNA duplexes. These analogs can be made stable in cellular mediaby a derivatization at their termini with simple L sugars. The use ofANAs in therapy is discussed, for example, in Damha et al., NucleosidesNucleotides & Nucleic Acids 20: 429-440, 2001.

Delivery of Nucleobase Oligomers

We demonstrate herein that naked oligonucleotides are capable onentering tumor cells and inhibiting IAP expression. Nonetheless, it maybe desirable to utilize a formulation that aids in the delivery ofoligonucleotides or other nucleobase oligomers to cells (see, e.g., U.S.Pat. Nos. 5,656,611, 5,753,613, 5,785,992, 6,120,798, 6,221,959,6,346,613, and 6,353,055, each of which is hereby incorporated byreference).

Ribozymes

Catalytic RNA molecules or ribozymes that include an antisense IAPsequence of the present invention can be used to inhibit expression ofan IAP polynucleotide in vivo. The inclusion of ribozyme sequenceswithin antisense RNAs confers RNA-cleaving activity upon them, therebyincreasing the activity of the constructs. The design and use of targetRNA-specific ribozymes is described in Haseloff et al., Nature334:585-591. 1988, and U.S. Patent Application Publication No.2003/0003469 A1, each of which is incorporated by reference.

Accordingly, the invention also features a catalytic RNA molecule thatincludes, in the binding arm, an antisense RNA having between eight andnineteen consecutive nucleobases corresponding to a sequence of any oneof Tables 1, 2, 6, and 7. In preferred embodiments of this invention,the catalytic nucleic acid molecule is formed in a hammerhead or hairpinmotif, but may also be formed in the motif of a hepatitis delta virus,group I intron or RNaseP RNA (in association with an RNA guide sequence)or Neurospora VS RNA. Examples of such hammerhead motifs are describedby Rossi et al., Aids Research and Human Retroviruses, 8:183, 1992.Example of hairpin motifs are described by Hampel et al., “RNA Catalystfor Cleaving Specific RNA Sequences,” filed Sep. 20, 1989, which is acontinuation-in-part of U.S. Ser. No. 07/247,100 filed Sept. 20, 1988,Hampel and Tritz, Biochemistry, 28:4929, 1989, and Hampel et al.,Nucleic Acids Research, 18: 299, 1990. An example of the hepatitis deltavirus motif is described by Perrotta and Been, Biochemistry, 31:16,1992. The RNaseP motif is described by Guernier-Takada et al., Cell,35:849, 1983. The Neurospora VS RNA ribozyme motif is described byCollins et al. (Saville and Collins, Cell 61:685-696, 1990; Saville andCollins, Proc. Natl. Acad. Sci. USA 88:8826-8830, 1991; Collins andOlive, Biochemistry 32:2795-2799, 1993). These specific motifs are notlimiting in the invention and those skilled in the art will recognizethat all that is important in an enzymatic nucleic acid molecule of thisinvention is that it has a specific substrate binding site which iscomplementary to one or more of the target gene RNA regions, and that ithave nucleotide sequences within or surrounding that substrate bindingsite which impart an RNA cleaving activity to the molecule.

RNA Interference

The nucleobase oligomers of the present invention may be employed indouble-stranded RNAs for RNA interference (RNAi)-mediated knock-down ofIAP expression. RNAi is a method for decreasing the cellular expressionof specific proteins of interest (reviewed in Tuschl, Chembiochem2:239-245, 2001; Sharp, Genes & Devel. 15:485-490, 2000; Hutvagner andZamore, Curr. Opin. Genet. Devel. 12:225-232, 2002; and Hannon, Nature418:244-251, 2002). In RNAi, gene silencing is typically triggeredpost-transcriptionally by the presence of double-stranded RNA (dsRNA) ina cell. This dsRNA is processed intracellularly into shorter piecescalled small interfering RNAs (siRNAs). The introduction of siRNAs intocells either by transfection of dsRNAs or through expression of siRNAsusing a plasmid-based expression system is increasingly being used tocreate loss-of-function phenotypes in mammalian cells.

In one embodiment of the invention, double-stranded RNA (dsRNA) moleculeis made that includes between eight and nineteen consecutive nucleobasesof a nucleobase oligomer of the invention. The dsRNA can be two distinctstrands of RNA that have duplexed, or a single RNA strand that hasself-duplexed (small hairpin (sh)RNA). Typically, dsRNAs are about 21 or22 base pairs, but may be shorter or longer (up to about 29 nucleobases)if desired. dsRNA can be made using standard techniques (e.g., chemicalsynthesis or in vitro transcription). Kits are available, for example,from Ambion (Austin, Tex.) and Epicentre (Madison, Wis.). Methods forexpressing dsRNA in mammalian cells are described in Brummelkamp et al.Science 296:550-553, 2002; Paddison et al. Genes & Devel. 16:948-958,2002. Paul et al. Nature Biotechnol. 20:505-508, 2002; Sui et al. Proc.Natl. Acad. Sci. USA 99:5515-5520, 2002; Yu et al. Proc. Natl. Acad.Sci. USA 99:6047-6052, 2002; Miyagishi et al. Nature Biotechnol.20:497-500, 2002; and Lee et al. Nature Biotechnol. 20:500-505 2002,,each of which is hereby incorporated by reference.

Small hairpin RNAs consist of a stem-loop structure with optional 3′UU-overhangs. While there may be variation, stems can range from 21 to31 bp (desirably 25 to 29 bp), and the loops can range from 4 to 30 bp(desirably 4 to 23 bp). For expression of shRNAs within cells, plasmidvectors containing either the polymerase III HI -RNA or U6 promoter, acloning site for the stem-looped RNA insert, and a 4-5-thymidinetranscription termination signal can be employed. The Polymerase IIIpromoters generally have well-defined initiation and stop sites andtheir transcripts lack poly(A) tails. The termination signal for thesepromoters is defined by the polythymidine tract, and the transcript istypically cleaved after the second uridine. Cleavage at this positiongenerates a 3′ UU overhang in the expressed shRNA, which is similar tothe 3′ overhangs of synthetic siRNAs. Additional methods for expressingthe shRNA in mammalian cells are described in the references citedabove.

The following examples are to illustrate the invention. They are notmeant to limit the invention in any way.

EXAMPLE 1 Nucleobase Oligomer Selection

We selected 96 and 98, mostly non-overlapping, 19-mer nucleobasesequences for human XIAP and HIAP 1, respectively, based on theselection criteria listed below. In the case of XIAP, we selected 96sequences (each being 19 nucleobases in length) (SEQ ID NOs: I through96; Table 1), from a region approximately 1 kb upstream of the startcodon to approximately 1 kb downstream of the stop codon of the cDNAsequence. This blanketed approximately 50% of the coding region, andimmediate 5′ and 3′ UTR sequences (i.e., 96 19-mers span 1.8 kb ofsequence, while the targeted region is approximately 3.5 kb in length,comprised of a coding region of 1.5 kb plus 1 kb at either side of UTRsequences). TABLE 1 SEQ XIAP down- XIAP down- XIAP down- ID regulationregulation regulation NO: Code Nucleobase Sequence T24 RNA T24 proteinH460 RNA 1 A1 AAAATTCTAAGTACCTGCA — — 48% 2 B1 TCTAGAGGGTGGCTCAGGA — —66% 3 C1 CAGATATATATGTAACACT — — 66% 4 D1 TGAGAGCCCTTTTTTTGTT — — 75% 5E1 AGTATGAAATATTTCTGAT — — 69% 6 F1 ATTGGTTCCAATGTGTTCT — — 81% 7 G1TTAGCAAAATATGTTTTAA — — 33% 8 H1 TGAATTAATTTTTAATATG — — 13% 9 A2ATTCAAGGCATCAAAGTTG — — 58% 10 B2 GTCAAATCATTAATTAGGA — — 55% 11 C2AATATGTAAACTGTGATGC 36% 45% 70% 12 D2 GCAGAATAAAACTAATAAT — — 39% 13 E2GAAAGTAATATTTAAGCAG 54% 51% 60% 14 F2 TTACCACATCATTCAAGTC — — 34% 15 G2CTAAATACTAGAGTTCGAC — — 55% 16 H2 ACACGACCGCTAAGAAACA — — 46% 17 A3TATCCACTTATGACATAAA — — 27% 18 B3 GTTATAGGAGCTAACAAAT — — 34% 19 C3AATGTGAAACACAAGCAAC — — 43% 20 D3 ACATTATATTAGGAAATCC — — 30% 21 E3CTTGTCCACCTTTTCTAAA 53% 64% 55% 22 F3 ATCTTCTCTTGAAAATAGG 44% 53% — 23G3 CCTTCAAAACTGTTAAAAG — — — 24 H3 ATGTCTGCAGGTACACAAG — — — 25 A4ATCTATTAAACTCTTCTAC — — — 26 B4 ACAGGACTACCACTTGGAA — — 76% 27 C4TGCCAGTGTTGATGCTGAA 28% 56% 77% 28 D4 GTATAAAGAAACCCTGCTC 12% 43% 51% 29E4 CGCACGGTATCTCCTTCAC 47% 34% 51% 30 F4 CTACAGCTGCATGACAACT 33% 43% —31 G4 GCTGAGTCTCCATATTGCC 34% 48% 51% 32 H4 ATACTTTCCTGTGTCTTCC — — — 33A5 GATAAATCTGCAATTTGGG — — — 34 B5 TTGTAGACTGCGTGGCACT — — 61% 35 C5ACCATTCTGGATACCAGAA 71% 54% — 36 D5 AGTTTTCAACTTTGTAGTG 39% 33% — 37 E5ATGATCTCTGCTTCCCAGA — — 46% 38 F5 AGATGGCCTGTCTAAGGCA — — — 39 G5AGTTCTCAAAAGATAGTCT — — 30% 40 H5 GTGTCTGATATATCTACAA — — 39% 41 A6TCGGGTATATGGTGTCTGA — — 72% 42 B6 CAGGGTTCCTCGGGTATAT 51% 47% — 43 C6GCTTCTTCACAATACATGG — — — 44 D6 GGCGAGTTCTGAAAGGACT — — 60% 45 E6GCTAACTCTCTTGGGGTTA — — — 46 F6 GTGTAGTAGAGTCCAGCAC 34% 39% — 47 G6AAGCACTGCACTTGGTCAC — — 69% 48 H6 TTCAGTTTTCCACCACAAC — — 68% 49 A7ACGATCACAAGGTTCCCAA — — — 50 B7 TCGCCTGTGTTCTGACCAG — — — 51 C7CCGGCCCAAAACAAAGAAG — — 72% 52 D7 GATTCACTTCGAATATTAA 56% 88% 46% 53 E7TATCAGAACTCACAGCATC — — — 54 F7 GGAAGATTTGTTGAATTTG — — 69% 55 G7TCTGCCATGGATGGATTTC — — 41% 56 H7 AAGTAAAGATCCGTGCTTC — — 63% 57 A8CTGAGTATATCCATGTCCC — — — 58 B8 GCAAGCTGCTCCTTGTTAA — — — 59 C8AAAGCATAAAATCCAGCTC — — 16% 60 D8 GAAAGCACTTTACTTTATC 38% 26% 49% 61 H8ACTGGGCTTCCAATCAGTT — — — 62 E8 GTTGTTCCCAAGGGTCTTC 72% 56% 44% 63 F8ACCCTGGATACCATTTAGC — — 47% 64 G8 TGTTCTAACAGATATTTGC — — 49% 65 A9TATATATTCTTGTCCCTTC — — 62% 66 B9 AGTTAAATGAATATTGTTT — — 38% 67 C9GACACTCCTCAAGTGAATG — — — 68 D9 TTTCTCAGTAGTTCTTACC — — 39% 69 E9GTTAGTGATGGTGTTTTCT — — 43% 70 F9 AGATGGTATCATCAATTCT — — 19% 71 G9TGTACCATAGGATTTTGGA — — — 72 H9 CCCGATTCGTATAGCTTCT — — — 73 A10ATTATTTTCTTAATGTCCT — — 29% 74 B10 CAAGTGATTTATAGTTGCT — — — 75 C10TAGATCTGCAACCAGAACC — — 53% 76 D10 GATCTTGCATACTGTCTTT — — 55% 77 E10CCTTAGCTGCTCTTCAGTA — — — 78 F10 AAGCTTCTCCTCTTGCAGG — — 51% 79 G10ATATTTCTATCCATACAGA — — 56% 80 H10 CTAGATGTCCACAAGGAAC — — — 81 A11AGCACATTGTTTACAAGTG — — 68% 82 B11 AGCACATGGGACACTTGTC — — 63% 83 C11CTTGAAAGTAATGACTGTG — — 52% 84 D11 CCTACTATAGAGTTAGATT — — — 85 E11ATTCAATCAGGGTAATAAG — — 48% 86 F11 AAGTCAGTTCACATCACAC — — 64% 87 G11CAGTAAAAAAAATGGATAA — — 33% 88 H11 TTCAGTTATAGTATGATGC — — — 89 A12TACACTTAGAAATTAAATC — — 46% 90 B12 TCTCTATCTTTCCACCAGC — — — 91 C12AGAATCCTAAAACACAACA — — — 92 D12 ATTCGCACAAGTACGTGTT — — 77% 93 E12TGTCAGTACATGTTGGCTC — — 74% 94 F12 ACATAGTGTTTTGCCACTT — — 74% 95 G12CTTTGATCTGGCTCAGACT — — 76% 96 H12 GAAACCAGATTTAACAGTT — — 52%

Note that in any of the foregoing nucleobase oligomers, or any othernucleobase oligomers described herein, each nucleobase may independentlybe a DNA residue or RNA residue, such as a 2′-O-methyl or2′-O-methoxyelthyl RNA residue. For example, the nucleobase sequence ofSEQ ID NO: 3 may be, for example, 5′-CAGATATATATG TAACACT-3′,5′-CAGATATATATGTAACACU-3′, or 5′-mCmAGATATATATGTA ACAmCmU-3′ (wherein mXrepresents a 2′-O-methyl X residue). Additional modified nucleobases areknown in the art. The linkages may be phosphodiester (PO),phosphorothioate (PS), or methylphosphonate (MP) linkages, or may have amixed backbone (MB). The backbone may be any suitable backbone thatallows hybridization of the nucleobase oligomer to the target IAPpolynucleotide. Exemplary backbones are described herein. In otherembodiments, the nucleobase oligomers include acridine-protectedlinkages, cholesteryl or psoralen components, C5-propynyl pyrimidines,or C5-methyl pyrimidines. Suitable modifications to the nucleobaseoligomers of the invention include those described above, as well asthose in U.S. Patent Application Publication No. U.S. 2002/0128216 A1,hereby incorporated by reference.

Examples of nucleobase oligomers are provided in Table 2, below (whereinmX represents a 2′-O-methyl X RNA residue). TABLE 2 SEQ ID NO: 2 × 2 MBPO DE4 as mGmGTATCTCCTTCACCAGmUmA 97 DE4 rev mAmUGACCACTTCCTCTATmGmG 98δBC5 as mGmATACCAGAATTTmGmU 99 δBC5 rev mUmGTTTAAGACCATmAmG 100 mG4 asmGmCTGAGTCTCCATACTGmCmC 101 mG4 sm mGmGCTCTCTGCCCACTGAmAmU 102 3 × 3 MBPO F3 as mAmUmCTTCTCTTGAAAATmAmGmG 103 F3 scr mCmAmGAGATTTCATTTAAmCmGmU104 F3 mm mAmUmCTTGACTTGATTATmAmGmG 105 F3 rev mGmGmATAAAAGTTCTCTTmCmUmA106 E4 as mCmGmCACGGTATCTCCTTmCmAmC 107 E4 scr mCmUmACGCTCGCCATCGTmUmCmA108 E4 rev mCmAmCTTCCTCTATGGCAmCmGmC 109 E4 mm mCmGmCACCCTATCTGGTTmCmAmC110 G4 as mGmCmUGAGTCTCCATATTmGmCmC 111 G4 scr mGmGmCTCTTTCGCCACTGmAmAmU112 G4 rev mCmCmGTTATACCTCTGAGmUmCmG 113 G4 mm mGmCmUGACACTCCAATTTmGmcmC114 C5 as mAmCmCATTCTGGTAACCAmGmAmA 115 C5 scr mUmGmCCCAAGAATACTAGmUmCmA116 C5 mm mAmCmCATAGTGGATTGCAmGmAmA 117 C5 rev mAmAmGACCATAGGTCTTAmCmCmA118 D7 as mGmAmUTCACTTCTTCGAATATmUmAmA 119 D7 scrmUmGmAAATGTAAATCATCmUmUmC 120 D7 mm mGmAmUTCTGTTCGATAATmUmAMA 121 D7 revmAmAmUTATAAGCTTCACTmUmAmG 122 Phosphorothioate PS-G4 asGCTGAGTCTCCATATTGCC 123 PS-G4 sm GGCTCTTTGCCCACTGAAT 124 PS-C5 asACCATTCTGGATACCAGAA 125 PS-C5 rev AAGACCATAGGTCTTACCA 126 PS-F3 asATCTTCTCTTGAAAATAGG 127 PS-F3 rev GGATAAAAGTTCTCTTCTA 128 PS-DE4 asGGTATCTCCTTCACCAGTA 129 PS-DE4 rev ATGACCACTTCCTCTATGG 130 PS-BC5 asTCTGGATACCAGAATTTGT 131 PS-BC5 rev TGTTTAAGACCATAGGTCT 132 PS-AB6 asGGGTTCCTCGGGTATATGG 133 PS-AB6 rs GGTATATGGCGTCCTTGGG 134 PS-D7 asGATTCACTTCGAATATTAA 135 PS-D7 rs AATTATAACGTTCACTTAG 136 Penetratin F3as ATCTTCTCTTGAAAATAGG 137 G4 as GCTGAGTCTCCATATTGCC 138 D7 asGATTCACTTCGAATATTAA 139 C5 cs TGCCCAAGAATACTAGTCA 140 4 × 4 MBO PS(phosphorothioate linkages throughout) G4 as mGmCmUmGAGTCTCGATATmUmGmCmC141 G4 sm mGmGmCmUCTTTGCCCACTmGmAmAmU 142 DE4 asmGmGmUmATCTCCTTCACCmAmGmUmA 143 DE4 rev mAmUmGmACCACTTCCTCTmAmUmGmG 144E2 as mGmAmAmAGTAATATTTAAmGmCmAmG 145 E2 rm mGmAmGmCAATTTATAATGmAmAmAmG146 H2G as mAmCmCmGCTAAGAAACATmUmCmUmA 147 H2G rmmAmUmCmUTACAAAGAATCmCmGmCmA 148 A3 as mUmAmUmCCACTTATGACAmUmAmAmA 149 A3rev mAmAmAmUACAGTATTCACmCmUmAmU 150 FG8 as mUmGmCmACCCTGGATACCmAmUmUmU151 FG8 rm mUmUmUmACCATAGGTCCCmAmGmCmU 152 mG4 asmGmCmUmGAGTCTCCATACmUmGmCmC 153 mG4 sm mGmGmCmUCTCTGCCCACTmGmAmAmU 154F1 as mAmUmUmGGTTCCAATGTGmUmUmCmU 155 F1 rev mUmCmUmUGTGTAACCTTGmGmUmUmA156 B4 as mAmCmAmGGACTACCACTTmGmGmAmA 157 B4 revmAmAmGmGTTCACCATGAGmGmAmCmA 158 G6 as mAmAmGmCACTGCACTTGGmUmCmAmC 159 G6sm mCmAmCmTGGTTGACCTCAmCmAmAmG 160 E12 as mUmGmUmCAGTACATGTTGmGmCmUmC161 E12 sm mCmUmAmGGTTGTCCATGAmCmUmGmU 162Penetratin and its use in mediating entry of nucleobase oligomers intocells are described in PCT patent application No. FR 91/00444.

A similar identification approach was taken for designing nucleobaseoligomers against HIAP1. Initially, 98 19-mer nucleobase oligomers werechosen (SEQ ID NOs: 163-260; Table 3). Of these 98 nucleobase oligomerstargeted to the HIAP1 sequence, fifteen (SEQ ID NOs: 163-170, 173, 179,202, 222, 223, 247, and 259) were selected for further evaluation. Thesefifteen candidate nucleobase oligomers included four nucleobaseoligomers targeting the coding region (SEQ ID NOs: 202, 222, 223, and247), one nucleobase oligomer targeting the 3′ UTR (SEQ ID NO: 259),seven nucleobase oligomers targeting the 5′ UTR (SEQ ID NOs: 166-170,173, and 179; one of the seven nucleobase oligomers overlapped the startcodon), and three other oligonucleotides (SEQ ID NOs: 163-165) that weredesigned to target an intronic segment of the 5′ UTR. TABLE 3 SEQ ID NO:Code Nucleobase Oligomer Sequence 163 APO 1 TCATTTGAGCCTGGGAGGT 164 APO2 CGGAGGCTGAGGCAGGAGA 165 APO 3 GGTGTGGTGGTACGCGCCT 166 APO 4ACCCATGCACAAAACTACC 167 APO 5 AGAATGTGCCAGTAGGAGA 168 APO 6TCTCACAGACGTTGGGCTT 169 APO 7 CCAGTGGTTTGCAAGCATG 170 APO 8GAAATTTAGTGGCCAGGAA 171 AGAAATACACAATTGCACC 172 TACTGATACATTTTAAGGA 173APO 9 TTCAACATGGAGATTCTAA 174 ATTTCTATGCATTTAGAGT 175AATACTAGGCTGAAAAGCC 176 GGCTTTGCTTTTATCAGTT 177 TCTAGGGAGGTAGTTTTGT 178GGGAAGAAAAGGGACTAGC 179 APO 10 GTTCATAATGAAATGAATG 180ATAAGAATATGCTGTTTTC 181 TTCAAACGTGTTGGCGCTT 182 ATGACAAGTCGTATTTCAG 183AAGTGGAATACGTAGACAT 184 AGACAGGAACCCCAGCAGG 185 CGAGCAAGACTCCTTTCTG 186AGTGTAATAGAAACCAGCA 187 TGACCTTGTCATTCACACC 188 TTATCCAGCATCAGGCCAC 189ACTGTCTCCTCTTTTCCAG 190 TTTTATGCTTTTCAGTAGG 191 ACGAATCTGCAGCTAGGAT 192CAAGTTGTTAACGGAATTT 193 TAGGCTGAGAGGTAGCTTC 194 GTTACTGAAGAAGGAAAAG 195GAATGAGTGTGTGGAATGT 196 TGTTTTCTGTACCCGGAAG 197 GAGCGACGGAAATATCCAC 198TGATGGAGAGTTTGAATAA 199 GATTTGCTCTGGAGTTTAC 200 GGCAGAAAATTCTTGATTT 201GGACAGGGGTAGGAACTTC 202 APO 11 GCATTTTCGTTATTCATTG 203CTGAAAAGTAAGTAATCTG 204 GGCGACAGAAAAGTCAATG 205 CCACTCTGTCTCCAGGTCC 206CCACCACAGGCAAAGCAAG 207 TTCGGTTCCCAATTGCTCA 208 TTCTGACATAGCATTATCC 209TGGGAAAATGTCTCAGGTG 210 TATAAATGGGCATTTGGGA 211 TGTCTTGAAGCTGATTTTC 212GAAACTGTGTATCTTGAAG 213 TGTCTGCATGCTCAGATTA 214 GAATGTTTTAAAGCGGGCT 215CACTAGAGGGCCAGTTAAA 216 CCGCACTTGCAAGCTGCTC 217 CATCATCACTGTTACCCAC 218CCACCATCACAGCAAAAGC 219 TCCAGATTCCCAACACCTG 220 CCCATGGATCATCTCCAGA 221AACCACTTGGCATGTTGAA 222 APO 12 CAAGTACTCACACCTTGGA 223 APO 13CCTGTCCTTTAATTCTTAT 224 TGAACTTGACGGATGAACT 225 TAGATGAGGGTAACTGGCT 226TGGATAGCAGCTGTTCAAG 227 CATTTTCATCTCCTGGGCT 228 TGGATAATTGATGACTCTG 229GTCTTCTCCAGGTTCAAAA 230 TATTCATCATGATTGCATC 231 CATTTCCACGGCAGCATTA 232CCAGGCTTCTACTAAAGCC 233 GCTAGGATTTTTCTCTGAA 234 TCTATAATTCTCTCCAGTT 235ACACAAGATCATTGACTAG 236 TCTGCATTGAGTAAGTCTA 237 CTCTTCCCTTATTTCATCT 238TCCTCAGTTGCTCTTTCTC 239 GCCATTCTATTCTTCCGGA 240 AGTCAAATGTTGAAAAAGT 241CCAGGATTGGAATTACACA 242 ATTCCGGCAGTTAGTAGAC 243 TAACATCATGTTCTTGTTC 244GTCTGTGTCTTCTGTTTAA 245 TTCTCTTGCTTGTAAAGAC 246 CTAAAATCGTATCAATCAG 247APO 14 GGCTGCAATATTTCCTTTT 248 GAGAGTTTCTGAATACAGT 249ACAGCTTCAGCTTCTTGCA 250 AAATAAATGCTCATATAAC 251 GAAACATCTTCTGTGGGAA 252GTTCTTCCACTGGTAGATC 253 CTTCTTGTAGTCTCCGCAA 254 TTGTCCATACACACTTTAC 255AACCAAATTAGGATAAAAG 256 ATGTTCATATGGTTTAGAT 257 TAAGTTTTACTTCACTTAC 258ATGTTCCCGGTATTAGTAC 259 APO 15 GGGCTCAAGTAATTCTCTT 260GCCCAGGATGGATTCAAACNucleobase Oligomer Selection Criteria

The computer program OLIGO (previously distributed by NationalBiosciences Inc.) was used to select candidate nucleobase oligomersbased on the following criteria:

-   -   1) no more than 75% GC content, and no more than 75% AT content;    -   2) preferably no nucleobase oligomers with four or more        consecutive G residues (due to reported toxic effects, although        one was chosen as a toxicity control);    -   3) no nucleobase oligomers with the ability to form stable        dimers or hairpin structures; and    -   4) sequences around the translation start site are a preferred        region.        In addition, accessible regions of the target mRNA were        predicted with the help of the RNA secondary structure folding        program MFOLD (M. Zuker, D. H. Mathews & D. H. Turner,        Algorithms and Thermodynamics for RNA Secondary Structure        Prediction: A Practical Guide. In: RNA Biochemistry and        Biotechnology, J. Barciszewski & B. F. C. Clark, eds., NATO ASI        Series, Kluwer Academic Publishers, (1999). Sub-optimal folds        with a free energy value within 5% of the predicted most stable        fold of the mRNA were predicted using a window of 200 bases        within which a residue can find a complimentary base to form a        base pair bond. Open regions that did not form a base pair were        summed together with each suboptimal fold and areas that        consistently were predicted as open were considered more        accessible to the binding to nucleobase oligomers. Additional        nucleobase oligomer that only partially fulfilled some of the        above selection criteria were also chosen as possible candidates        if they recognized a predicted open region of the target mRNA.

EXAMPLE 2 Oligonucleotide Synthesis

The ability of nucleobase oligomers to inhibit IAP expression was testedusing oligonucleotides as exemplary nucleobase oligomers. Theoligonucleotides were synthesized by IDT (Integrated DNA Technologies,USA) as chimeric, second-generation oligonucleotides, consisting of acore of phosphodiester DNA residues flanked on either side by two2′-O-methyl RNA residues with a phosphorothioate linkage between theflanking RNA residues. The oligonucleotides were provided in a 96-wellplate, as well as matching tubes, with a minimum of 12 ODs of nucleobaseoligomer, which provided ample material for transfections (greater thana hundred assays in the 96-well format) when the detection method is asensitive method, such as TaqMan quantitative PCR, or an ELISA. Once thepositive hits were identified (see below), oligonucleotides werere-synthesized with three, instead of two, flanking RNA residues tofurther increase stability/nuclease resistance. In addition, forvalidation purposes, appropriate controls (such as scrambled, 4-basemismatch, and reverse polarity oligonucleotides) were synthesized forsome of the targets that yielded the highest activity.

EXAMPLE 3 Screening Assays and Optimization of Nucleobase Oligomers

Our approach to identifying nucleobase oligomers capable of inhibitingexpression of an IAP was to screen the above-described oligonucleotidelibraries for specific decreases (knock-down) of the RNA and/or proteinfor the specific IAP gene targeted. Any number of standard assays may beused to detect RNA and protein levels in cells. For example, RNA levelscan be measured using standard northern blot analysis or RT-PCRtechniques. Protein levels can be measured, for example, by standardwestern blot analyses or immunoprecipitation techniques. Alternatively,cells administered an antisense IAP nucleic acid may be examined forcell viability, according to methods described, for example, in U.S.Pat. Nos. 5,919,912, 6,156,535, and 6,133,437, incorporated herein byreference.

We used TaqMan quantitative PCR (described below) to assay for changesin mRNA levels after oligonucleotide treatment. We employed ELISA fordetermining XIAP protein levels and western blotting for determiningHIAP1 protein levels. Transfection conditions were optimized withLipofectamine plus or Lipofectamine 2000 (Life Technologies, Canada) onT24 bladder carcinoma cells or H460 non-small cell lung carcinoma cells,or lipofectin on SF-295 glioblastoma cells, using a fluorescein-taggedsense oligonucleotide (5′-mGmAGAAGATGACTGGTAAmCmA-3′; SEQ ID NO: 261)from XIAP spanning the start codon as a control. The results werevisualized and gauged by epi-fluorescence microscopy. In the case of T24cells, transfections were further optimized based on the ability of apublished oligonucleotide to downregulate survivin expression (Li etal., Nat. Cell Biol. 1:461-466, 1999) (5′-U/TGTGCTATTCTGTGAA U/TU/T-3′SEQ ID NO: 262). We optimized the transfection conditions based on theTaqMan results of survivin RNA knock-down detected with PCR primers andfluorescent probe, described in detail below. Optimal conditions foroligonucleotide uptake by the cells were found to be 940 nMoligonucleotide and 4 μL PLUS reagent and 0.8 μL Lipofectamine in atotal of 70 μL for three hours. We then applied these conditions toscreen for XIAP protein knock-down using the oligo library against T24cells.

HIAP1 knock-down was studied in SF-295 cells because these cells hadeasily detectable and discernable 70 kDa HIAP1 protein, while many celllines do not express high levels of the protein, or are notdistinguishable from the large amounts of the similarly sized 68 kDaHIAP2 protein.

Real-Time PCR

RNA was extracted from cells lysed in RLT buffer (QIAGEN, Valencia,Calif.), and purified using QIAGEN RNeasy columns/kits. Real-timequantitative PCR was performed on a Perkin-Elmer ABI 7700 Prism PCRmachine. RNA was reverse transcribed and amplified according to theTaqMan Universal PCR Master Mix protocol of PE Biosystems, using primersand probes designed to specifically recognize XIAP, HIAP1, survivin, orGAPDH. For human survivin, the forward primer was 5′-TCTGCTTCAAGGAGCTGGAA-3′ (SEQ ID NO: 263), the reverse primer was 5′-GAAAGGAAAGCGCAACCG-3′ (SEQ ID NO: 264), and the probe was 5′-(FAM)AGCCAGATGACGACCCCATAGAGGAACATA(TAMRA)-3′ (SEQ ID NO: 265). For human HIAP1, theforward primer was 5′-TGGAGATGATCCATGGGTTCA-3′ (SEQ ID NO: 266), thereverse primer was 5′-GAACTCCTGTCCTTTAATTCTTATCAAGT-3′ (SEQ ID NO: 267),and the probe was 5′-(FAM)CTCACACCTTGGAAACCACTTGGCATG (TAMRA)-3′ (SEQ IDNO: 268). For human XIAP, the forward primer was 5′-GGTGATAAAGTAAAGTGCTTTCACTGT-3′ (SEQ ID NO: 269), the reverse primer was5′-TCAGTAGTTCTTACCAGACACTCCTCAA-3′ (SEQ ID NO: 270), and the probe was5′-(FAM)CAACATGCTAAATGGTATCCAGGGTGCAAATATC(TAMRA)-3′ (SEQ ID NO: 271).For human GAPDH, the forward primer was 5′-GAAGGTGAAGG TCGGAGTC-3′ (SEQID NO: 272), the reverse primer was 5′-GAAGATGGTGATGG GATTC-3′ (SEQ IDNO: 273), and the probe was 5′-(JOE)CAAGCTTCCCGTTCTCA GCC(TAMRA)-3′ (SEQID NO: 274). FAM is 6-carboxyfluoroscein, JOE is6-carboxy-4,5-dichloro-2,7-dimethoxyfluoroscein, and TAMRA is6-carboxy-N,N,N′,N′-tetramethylrhodamine. FAM and JOE are 5′ reporterdyes, while TAMRA is a 3′ quencher dye.

Relative quantification of gene expression was performed as described inthe PE Biosystems manual using GAPDH as an internal standard. Thecomparative Ct (cycle threshold) method was used for relativequantitation of IAP mRNA levels compared to GAPDH mRNA levels. Briefly,real-time fluorescence measurements were taken at each PCR cycle and thethreshold cycle (Ct) value for each sample was calculated by determiningthe point at which fluorescence exceeded a threshold limit of 30 timesthe baseline standard deviation. The average baseline value and thebaseline SD are calculated starting from the third cycle baseline valueand stopping at the baseline value three cycles before the signal startsto exponentially rise. The PCR primers and/or probes for the specificIAPs were designed to span at least one exon-intron boundary separatedby 1 kb or more of genomic DNA, to reduce the possibility of amplifyingand detecting genomic DNA contamination. The specificity of the signal,and possible contamination from DNA, were verified by treating some RNAsamples with either DNase or RNase, prior to performing the reversetranscription and PCR reaction steps.

XIAP ELISA and HIAP1 Western Immunoblots

A standard colorimetric XIAP ELISA assay was performed using anaffinity-purified rabbit polyclonal antibody to XIAP as a captureantibody, and was detected with a XIAP monoclonal antibody (MBL, Japan)and a biotinylated anti-mouse Ig antibody and horseradishperoxidase-conjugated streptavidin and TMB substrate. Alternatively, apolyclonal XIAP or HIAP1 antibody may be used to measure XIAP or HIAP1protein levels, respectively.

HIAP1 was detected on a western immunoblot using an affinity-purifiedanti-rat HIAP1 rabbit polyclonal antibody as a primary antibody and wasdetected by ECL (Amersham) on X-ray film with a secondaryhorseradish-peroxidase-conjugated anti-rabbit Ig antibody and achemiluminescent substrate. The anti-HIAP1 polyclonal antibody is raisedagainst a GST-fusion of the rat HIAP1. This antibody cross-reacts withboth human and murine HIAP1 and HIAP2.

EXAMPLE 4 Antisense XIAP Oligonucleotides Decrease XIAP RNA andPolypeptide Expression

The XIAP synthetic library of 96 antisense oligonucleotides was firstscreened for decreases in XIAP protein levels. Specifically, T24 cells(1.5×10⁴ cells/well) were seeded in wells of a 96-well plate on day 1,and were cultured in antibiotic-free McCoy's medium for 24 hours. On day2, the cells were transfected with XIAP antisense oligonucleotides asdescribed above (oligonucleotides are labeled according to their platedposition, i.e., A1 to H12, and include two repeats, A13 and B13 thatcontain lyophilized DNA pellets that stuck to the sealing membrane).Briefly, the nucleobase oligomers were diluted in 10 μl/well ofserum-free, antibiotic-free McCoy's medium and then PLUS reagent wasadded. Lipofectamine was diluted in 10 μl/well of serum-free,antibiotic-free McCoy's medium, and both mixes were incubated for 15minutes at room temperature. The mixes were then combined and incubatedfor 15 minutes at room temperature.

In the meantime, the complete medium was removed from the cells and 50μl/well of serum-free, antibiotic-free medium was added to the cells.The transfection mixes were added to the well, and the cells wereincubated for three hours. Then 30 μl/well of serum-free,antibiotic-free medium and 100 μl/well of antibiotic-free completemedium, containing 20% fetal bovine serum were added to each well.

At day 3, XIAP RNA levels were measured using quantitative real-time PCRtechniques, as described above. At day 4, XIAP protein levels weremeasured by ELISA (FIGS. 1A, 1C, 1E, 1G, 1I, and 1K), and total cellularprotein was measured biochemically (FIGS. 1B, 1D, 1F, 1H, 1J, and 1L;used to normalize the XIAP protein levels). The results were compared toa mock transfection sample (treated with the transfection agent but nooligonucleotide DNA was added, and then processed as for the othersamples). Time course experiments determined that the optimal time forprotein knock-down to be around 12 to 24 hours.

The oligonucleotide library was also screened for decreases in RNAlevels, using TaqMan-specific PCR primers and fluorescent probes at theappropriate optimal time, using the primers and probes described above.Time course experiments determined mRNA to be optimally decreased at 6to 9 hours. These results agree well with the protein results.

The first screen (although performed at a sub-optimal time point whenXIAP levels are returning to normal, possibly due to an outgrowth ofnon-transfected cells) identified 16 antisense oligonucleotides (Table1: C2, E2, E3, F3, C4, D4, E4, F4, G4, C5, D5, B6, F6, D7, D8, F8) outof the 96 nucleobase oligomers tested that showed some decrease in XIAPprotein levels relative to total protein, compared to mock (nonucleobase oligomer) transfection levels (FIGS. 1A, 1C, 1E, 1G, 1I, and1K). Total protein was decreased for each of these 16 nucleobaseoligomers, which indicates a toxic or cytostatic effect of thesenucleobase oligomers (FIGS. 1B, 1D, 1F, 1H, 1J, 1L). Nucleobaseoligomers B9 and C9 showed a clear drop in total protein but no relativedrop in XIAP protein levels.

The 16 antisense nucleobase oligomers that showed some decrease inrelative XIAP protein levels compared to mock transfection, werere-tested alone or in combination, with one control nucleobase oligomer(D2) included, for their ability to knock-down XIAP protein at a moreoptimal time point (12 hours) based on the above described time coursestudies (FIG. 2B). These nucleobase oligomers were also examined fortheir ability to decrease XIAP mRNA levels at 12 hours, normalizedagainst GAPDH levels, and compared to mock transfection. Total proteinconcentrations at 12 hours were also determined (FIG. 2C).

There was a good correlation between the ability of a nucleobaseoligomer to decrease XIAP protein levels (FIG. 2B) with its ability todecrease XIAP MRNA levels (FIG. 2A). In addition, there is no major lossof total protein at this early time point, and the decrease in XIAP mRNAand protein precede the decrease in total protein that is seen at latertime points. The nucleobase oligomers that showed greater than 50% lossof XIAP protein or mRNA levels alone, or in a combination of twonucleobase oligomers added at a 1:1 ratio, were identified as the bestnucleobase oligomers and validated further. Of these 16oligonucleotides, ten (E2, E3, F3, E4, F4, G4, C5, B6, D7, F8) showed aconsistent ability to decrease XIAP protein or RNA levels by more than50%, depending on the transfection conditions used, or when used incombination (as for C5 and G4). Moreover, these 16 oligonucleotides thatdemonstrated antisense activity clustered in four different targetregions of the XIAP mRNA, with adjacent nucleobase oligomers showingsome knock-down activity. Little or no antisense activity was observedwith nucleobase oligomers that target sequences between these regions orislands of sensitivity. Presumably, these regions represent open areason the mRNA that are accessible to nucleobase oligomers inside the cell.Two nucleobase oligomers, E3 and F3, target XIAP just upstream of thestart codon in the intervening region between the IRES and thetranslation start site, and partially overlap the end of the IRESelement. C2, D2, and E2 target a XIAP region upstream of the minimalIRES element, providing further evidence that the minimal IRES region isa highly structured region of RNA that is not readily accessible tonucleobase oligomers in vivo. All the other nucleobase oligomers arecomplimentary to a portion of the coding region, including a cluster ofactivity at positions 856-916 of the XIAP sequence (E4, F4, and G4) andsmaller separate areas, as demonstrated by nucleobase oligomers C5 andD5, for example.

A portion of the 96 nucleobase oligomers depicted in Table 1 wererescreened for their ability to knock-down XIAP mRNA in NCI-H460 cellsat 9 hours post-transfection. The data are summarized in Table 4, below.TABLE 4 2 × 2 MBO XIAP RNA Std. Dev. Untrf. Co. 1.04 0.055 Mock Co. 1.010.006 G4 sm 0.97 0.071 DE4 rev 1.06 0.121 A1 as 0.46 0.01 B1 as 0.340.03 C1 as 0.3 0.04 D1 as 0.25 0.03 E1 as 0.31 0.01 F1 as 0.19 0.01 G1as 0.67 0.03 H1 as 0.87 0.03 A2 as 0.42 0.02 B2 as 0.45 0.03 C2 as 0.330.02 D2 as 0.66 0.01 E2 as 0.44 0.01 F2 as 0.64 0.02 G2 as 0.44 0.01 H2as 0.56 0.04 A3 as 0.71 0.03 B3 as 0.64 0.08 C3 as 0.55 0.04 D3 as 0.680.02 E3 as 0.48 0.02 B4 as 0.23 0.01 C4 as 0.22 0.04 D4 as 0.48 0.04 E4as 0.44 0.01 G4 as 0.48 0.02 B5 as 0.38 0.03 E5 as 0.52 0.05 G5 as 0.680.05 H5 as 0.59 0.09 A6 as 0.27 0 D6 as 0.39 0.03 G6 as 0.3 0.01 H6 as0.31 0.01 C7 as 0.27 0.02 D7 as 0.52 0.04 F7 as 0.3 0.04 G7 as 0.66 0.04H7 as 0.49 0.01 C8 as 1.01 0.08 D8 as 0.55 0.04 F8 as 0.62 0 G8 as 0.640.06 H8 as 0.61 0.06 A9 as 0.46 0.02 B9 as 0.74 0.07 D9 as 0.73 0.04 E9as 0.69 0.06 F9 as 0.97 0.15 A10 as 0.85 0.04 C10 as 0.56 0.01 D10 as0.54 0.01 F10 as 0.64 0 G10 as 0.49 0 A11 as 0.36 0.03 B11 as 0.39 0.02C11 as 0.44 0.03 E11 as 0.52 0.04 F11 as 0.36 0.05 G11 as 0.67 0.02 A12as 0.54 0.03 D12 as 0.23 0.05 E12 as 0.26 0.01 F12 as 0.26 0.03 G12 as0.24 0.05 H12 as 0.48 0.06We also determined whether 4×4 MBOs (all PS, DNA residues flanked onboth sides by four 2′-O-methyl RNA residues) were capable ofknocking-down XIAP protein in H460 cells. As shown in FIGS. 3 and 4, 4×4MBs of E12 and another oligonucleotide, FG8, were effective in amountsas low as 31 nM.

EXAMPLE 5 XIAP Antisense Nucleobase Oligomers Increase Cytotoxicity andChemosensitization

We investigated if XIAP antisense nucleobase oligomers couldchemosensitize the highly drug resistant T24 cells to traditionalchemotherapeutic agents, such as adriamycin or cisplatin. Antisenseoligonucleotides were chosen to represent some of the different XIAPtarget regions and tested for their cytotoxic effects, alone or incombination with other oligonucleotides or drugs. Five XIAP antisenseoligonucleotides were tested for their ability to kill or chemosensitizeT24 bladder carcinoma cells, and were compared to the effects of threecorresponding scrambled control oligonucleotides.

T24 cells were transfected with XIAP antisense oligonucleotides,scrambled oligonucleotides, no oligonucleotides (mock transfected), orwere left untreated. The cells were tested for viability 20 hours aftertransfection (with the exception of the untreated control) using theWST-1 tetrazolium dye assay in which WST-1 tetrazolium dye is reduced toa colored formazan product in metabolically active cells (FIG. 5A).

The occurrence of cytoxicity induced by oligonucleotide E4 was examinedby visually inspecting T24 cells that were left untreated, mocktransfected, or transfected with E4, E4 scrambled, E4 reverse polarity,or E4 mismatched oligonucleotides. Twenty hours after transfection, thecells were examined for morphology (FIG. 5D). Only the cell transfectedwith antisense E4 oligonucleotides showed signs of toxicity.

To examine the effects of the nucleobase oligomers on thechemosensitization of the T24 cells to cisplatin or adriamycin,oligonucleotides were tested for their ability to further kill T24 cellsin the presence of a fixed dose of adriamycin (0.5 μg/ml). Cells werefirst transfected with a oligonucleotide, then adriamycin was added foranother 20 hours. Viability was measured by WST-1 at the end of the20-hour drug treatment (FIG. 5B). Results are shown in FIG. 5C aspercentage viability compared to nucleobase oligomer treatment alone.

All five nucleobase oligomers tested (F3, E4, G4, C5, D7) as well ascombinations of E4+C5 and G4+C5, killed the T24 cells, leaving only10-15% surviving cells after 24 hours, as compared to the mock (nooligonucleotide) transfected cells, or to cells transfected with threecorresponding scrambled controls to F3 (5′-mCmAmGAGATTTCATTTAAmCmGmU-3′; SEQ ID NO: 275), E4 (5′-mCmUmACGCTCGCCATCGTm UmCmA-3′;SEQ ID NO: 276) and C5 (5′-mUmGmCCCAAGAATACTAGmUmC mA-3′; SEQ ID NO:277)(FIGS. 5A and 5C). Therefore, the toxicity is sequence-specific tothose nucleobase oligomers that reduce XIAP levels, and not to anon-sequence specific toxicity due to nucleobase oligomers thischemistry in general. This cytotoxicity may result from the combinedeffect of XIAP protein knock-down (and the expected loss ofanti-apoptotic protection afforded by XIAP) and the cytotoxicity of thetransfection itself.

The addition of a fixed dose of adriamycin or cisplatin at the end ofthe three hour transfection period resulted in a further decrease insurvival for some of the tested oligonucleotides, a further 40% drop insurvival after 20 hours for nucleobase oligomers F3, D7 and G4+C5combination (FIG. 5B), compared to their correspondingoligonucleotide-treated values (FIG. 5C). The values in FIG. 5B(oligonucleotide plus drug) are compared to the values ofoligonucleotide alone in FIG. 5C, which is set a 100% for each ODN. Onlythe results for adriamycin chemosensitization are shown; similar resultswere obtained when the cells were chemosensitized with cisplatin. At thefixed doses used, the mock and scrambled control transfections did notshow any increased loss of survival when either treated with adriamycin(FIG. 5B). Chemosensitization is only seen when XL1P levels aredecreased by a specific antisense oligonucleotide.

EXAMPLE 6 Down-Regulating Effects of Antisense Oligonucleotides on XIAPmRNA in H460 Cells

By using real-time PCR, antisense oligonucleotides (2×2 MBO, composed oftwo flanking 2′-O-methyl RNA residues at either end withphosphorothioate linkages, and a central core of 15 phosphodiester DNAresidues) were examined for their effects on XIAP mRNA in H460 cells. Inthis configuration, nucleobase oligomers F3, G4, C5, AB6 and DE4 reducedthe mRNA level by 50-70%, compared to untreated control, while D7 ASnucleobase oligomers reduced the mRNA level by 30% (FIG. 6). Incontrast, control nucleobase oligomers and transfectant agent alone(LFA) each only reduced the mRNA level to less than 20% of untreatedcontrol (FIG. 6). Nucleobase oligomers F3, G4 and C5 were selected forfurther study in vitro and in vivo. Additional knockdown of XIAP mRNAobserved by TaqMan analysis is depicted in FIGS. 7 and 8.

EXAMPLE 7 Down-Regulating Effects of Antisense Oligonucleotides on XIAPProtein

We characterized the potency of nucleobase oligomers F3, G4 and C5 inoligonucleotide configuration on the XIAP protein expression by westernblot analysis (FIGS. 9, 10A, and 10B). G4 AS oligonucleotides exhibitedthe strongest down-regulating effect on XIAP protein, reducing XIAPprotein levels by 62% at 24 h after the end of transfection at aconcentration of 1.2 μM (FIGS. 10A and 10B). F3 AS oligonucleotides at1.2 μM reduced XIAP protein level by 50%, while C5 AS oligonucleotidesdid not show sequence specific effects compared to its control (FIG.10B). In additional studies, E12 and FG8 AS oligonucleotidessignificantly reduced XIAP protein levels (FIG. 9).

EXAMPLE 8 Induction of Apoptosis by XIAP AS Oligonucleotides

Having demonstrated that XIAP AS nucleobase oligomers were capable ofreducing viability of H460 cells and T24 bladder carcinoma cells after,we determined whether the observed cell death was due to the inductionof apoptosis. As shown in FIG. 11A, H460 cells treated with F3 or G4 ASoligonucleotides at 1.2 μM activated and degraded pro-caspase-3 proteinwith a reduction of 40% or 60% of protein levels, respectively, comparedto untreated control cells. PARP was also to its predictedcaspase-3-generated fragment (FIG. 11A). In contrast, F3 and G4 SColigonucleotide controls at 1.2 μM did not have any effect on caspase-3or PARP protein expression (FIG. 11A). The ratio of Bcl-2:Bax wasunchanged in H460 cells treated with. F3 and G4 AS oligonucleotides andtheir respective controls at 1.2 μM. Flow cytometry was used to detectthe hypo-diploid DNA content in H460 cells treated with G4 ASoligonucleotides and stained with PI (FIG. 12A). When H460 cells weretreated with G4 AS oligonucleotides or scrambled controloligonucleotides at 1.2 μM, the hypo-diploid DNA content of cells was40.8 and 22. 1%, respectively, compared to 16.6% for untreated controlcells. DAPI staining was used to detect the nuclear morphologicalchanges of the H460 cells treated with G4 AS oligonucleotides orscrambled control oligonucleotides at 1.2 μM. As shown in FIG. 12B,cells treated with G4 AS oligonucleotides underwent morphologicalchanges characteristic of apoptosis, including chromatin condensationand nuclear DNA fragmentation. Few cells showed these morphologicalchanges in G4 SC-treated control cells.

EXAMPLE 9 Inhibition of Cell Growth and Sensitization of H460 Cells toAnticancer Agents by AS Oligonucleotides

To analyze biological effects of nucleobase oligomers associated withdown-regulation of XIAP expression and apoptosis, the growth of H460cells treated with G4 AS oligonucleotides was investigated by MTT assay.Forty-eight hours after the transfection, G4 AS oligonucleotides hadreduced H460 cell growth in a dose-dependent manner, exhibiting a 55%reduction relative to untreated control levels at 1.2 μM (FIG. 13A). Incontrast, the growth-inhibitory effect of G4 SC oligonucleotides, ortransfectant agent alone, was comparatively low, only less than 10% oftheir untreated control.

To investigate whether down-regulation of XIAP expression has thepotential to sensitize H460 cells to chemotherapy, combinationtreatments using G4 AS oligonucleotides and one of the followinganticancer drugs: doxorubicin (DOX), taxol, vinorelbine (VNB) andetoposide (Etop) were performed. FIG. 13B demonstrates that each of thecombinations resulted in at least an additive cytotoxic effect on thecell death, compared to treatment with either G4 AS oligonucleotides orthe anticancer drugs alone.

EXAMPLE 10 Antitumor Efficacy of G4 AS Oligonucleotides on 11460 andLCC6 Tumor Xenografts

We first determined whether intra-tumoral injection of XIAP antisense2×2-MBOs into SCID-RAG2 mice carrying sub-cutaneous H460 human lungcarcinoma xenografts reduced the amount of tumor growth. Treatmentstarted 14 days after tumor ell inoculation (s.c. shoulder injection of106 cells) by injecting MBOs (50 μg 2′-O-methyl RNA oligonucleotides perg tumor) into the palpable tumor mass three times per week for twoweeks. Vinorelbine (VNB; also referred to as navelbine (NVB) (15 mg/kgi.p.) was injected on days 17 and 24. Tumor size was measured withcalipers three times per week. At the end of the treatment period (day24), the mean relative tumor growth of mice treated with a combinationof C5+G4 AS MBOs and VNB was ˜70% reduced compared to those treated withscrambled control MBOs and VNB. Treatments with C5 AS MBO and VNBresulted in a ˜60% reduction of tumor size, compared to scrambledcontrol (FIG. 14).

Initial systemic PS-oligonucleotide studies were designed without anychemotherapeutic agents. SCID-RAG2 mice were inoculated with H460 humanlung carcinoma cells (s.c. shoulder injection of 10⁶ cells) andtreatments with G4 and F3 AS PS-oligonucleotides, as well as a scrambledcontrol, were initiated three days after tumor inoculation. Nucleobaseoligomer injections were administered i.p. at 12.5 mg/kg three times aweek for three weeks. At the end of the treatment period, mean tumorsizes in the groups treated with either G4 or F3 AS oligonucleotideswere 50% smaller than in the group treated with a scrambled controloligonucleotides (FIG. 15). The same treatment protocol was tested onfemale SCID-RAG2 mice inoculated orthotopically with MDA-MB-435/LCC6human breast carcinoma cells. Two weeks after the last treatment (day35) tumor volumes of mice treated with F3, C5 or G4 AS oligonucleotideswere 70%, 60%, and 45%, respectively, smaller than vehicle controls(FIG. 16).

We conducted additional examination of the antitumor effects of G4 ASoligonucleotides in SCID-RAG2 mice bearing xenografts of H460 humannon-small-cell lung tumors implanted subcutaneously. Saline-treatedcontrol tumors grew reproducibly to a size of 0.75 cm³ withinapproximately 24 days (FIG. 17). Oligonucleotide treatments wereinitiated three days after tumor cell inoculation. G4 ASoligonucleotides (5 to 15 mg/kg) were administered using a treatmentschedule of i.p. injections given once a day on days 3-7, 10-14, and17-21. The treatment with 5 or 15 mg/kg G4 AS oligonucleotides greatlydelayed tumor growth: on day 24 mean tumor sizes were 0.75, 0.45 and0.29 cm³ in control, 5 and 15 mg/kg treated groups, respectively (FIG.18A). There was a dose-dependent inhibition of tumor growth. Tumor sizein mice treated with 15 mg/kg G4 AS oligonucleotides was significantlysmaller than in control groups, and represented 39% of control meantumor size. In contrast, administration of G4 SC oligonucleotides at 15mg/kg provided no therapeutic activity (FIG. 17). None of the micetreated with oligonucleotides displayed any signs of toxicities, andboth doses of oligonucleotides were well tolerated. A dose of 15 mg/kgwas selected for the future combination treatment regimens withanticancer drugs.

EXAMPLE 11 XIAP Expression is Reduced in H460 Tumors Treated With G4 ASOligonucleotides

To correlate the tumor growth inhibitory effects of G4 ASoligonucleotides with XIAP protein expression, we examined the changesin XIAP expression at the end of the in vivo treatment with 15 mg/kg ofG4 AS and SC oligonucleotides. At day 21 or 24 post-tumor inoculationwhen tumors reached 1 cm in size (FIG. 17), tumors were harvested andlysates from tumor homogenates were used for western blot analysis. XIAPand β-actin antibodies against the human protein were used, allowing fordetermination of human XIAP levels obtained from tumor cells specimenswithout contamination from XIAP derived from mouse cells. XIAP proteinlevels in tumors treated with G4 AS oligonucleotides were significantlyreduced to approximately 85% of control tumors (P<0.005) (FIGS. 18A and18B). Tumors treated with G4 SC oligonucleotides were reduced in size by24% of control tumors. These results indicated that inhibition of H460tumor growth by G4 AS oligonucleotides correlated with thedown-regulation of XIAP protein expression.

EXAMPLE 12 Histopathology of Tumor Specimens

To evaluate whether XIAP AS oligonucleotide administration results indirect tumor cell kill, we examined the histology of tumors aftertreatment both for morphology and ubiquitin immunostaining (FIGS. 19Aand 19B). At day 21 or 24 post-tumor inoculation, tumors treated with 15mg/kg of G4 AS oligonucleotides, SC oligonucleotides, or saline controlwere excised, sectioned, and stained with hematoxylin and eosin. Theresults demonstrate that tumors in animals administered given XIAP ASoligonucleotides treatment contained an increased number of dead cells,identified morphologically by their amorphous shape and condensednuclear material (FIG. 19A).

The degradation of proteins is largely ubiquitin-proteasome-dependent;increased ubiquitin expression has been observed during apoptosis. Thus,we examined the ubiquitin expression in the tumors sections used forhematoxylin and eosin staining. As shown in FIG. 19B, tumors in miceadministered XIAP AS oligonucleotides displayed more intenseimmunohistochemical staining, relative to tumors in control or SCODN-treated mice. These data indicate that there is more free ubiquitinand/or ubiquitinated-protein in XIAP AS nucleobaseoligonucleotide-treated tumor cells than in control tumors.

EXAMPLE 13 Combined Treatment of G4 AS Oligonucleotides With Vinorelbine

To evaluate whether combined treatments of G4 AS nucleobase oligomersand vinorelbine (VNB), a chemotherapeutic agent used for lung cancertreatment, may result in any cooperative effects, we compared thetherapeutic efficacy of VNB in the presence and absence of G4 ASoligonucleotides or 64 SC oligonucleotides. Treatment regimens wereinitiated on day 3 after tumor inoculation. FIG. 20A shows the in vivoefficacy results for 5 mg/kg and 10 mg/kg doses of VNB given to H460tumor-bearing mice and compared with saline controls. Each of the tworegimens induced significant tumor growth suppression in adose-dependent manner without showing significant signs of undesirabletoxicity (i.e., body weight loss). When administration of G4 ASoligonucleotides (15 mg/kg) was combined with VNB (5 mg/kg) for thetreatment of H460 tumors, even more pronounced delay of H460 tumorgrowth was observed compared to either treatment administrated alone(FIG. 20B). Again, the mice did not show any significant signs oftoxicity (i.e., body weight loss). The mean tumor sizes in mice treatedwith 5 mg/kg VNB in the presence or absence of G4 AS. or SColigonucleotides were compared on day 29 (FIGS. 20A and 20B). Theaverage tumor size in the group of VNB and G4 AS oligonucleotides was0.22±0.03 cm³, which was significantly smaller than the average tumorsize in animals treated with 5 mg/kg VNB alone or with a combination ofVNB G4 SC oligonucleotides (0.59±0.04 and 0.48±0.05 cm³, respectively).

Methods

The results obtained in Examples 5-13 were obtained using the followingmethods.

Oligonucleotide Synthesis

A library of over 96 non-overlapping chimeric, or mixed-backbone (MBO),19-mer antisense oligonucleotides was synthesized as 2×2 MBOoligonucleotides, composed of two flanking 2′-O-methyl RNA residues ateither end with phosphorothioate linkages, and a central core of 15phosphodiester DNA residues. Each final product was desalted by SephadexG-25 chromatography (IDT Inc., Coralville, Iowa). This chimeric wingmerconfiguration, and mix of phosphorothioate and phosphodiester linkages(referred to as 2×2 PS/PO), provided adequate stability while alsoreducing non-specific toxicity associated with phosphorothioateresidues. Fully phosphorothioated non-chimeric (DNA) antisenseoligonucleotides for in vivo and in vitro studies were synthesized byTrilink Biotech and purified by RP-HPLC.

Antisense Oligonucleotide Screening

T24 bladder carcinoma cells, transfected with 1-1.2 μMoligonucleotide-lipofectin complexes for 24-48 hours, were assessed todetermine the ability of each oligonucleotide to knock-down XIAPprotein. Positive hits were reconfirmed for their ability to knock-down(i) XIAP protein levels at 12-18 hours of transfection by westernanalysis, and (ii) XIAP mRNA levels at 6-9 hours of transfection byquantitative RT-PCR (see below) in T24 bladder carcinoma cells and H460lung carcinoma cells. Candidate oligonucleotides were identified andtested further. Identified 2×2 PS/PO chimeric oligonucleotides showed adose-dependent ability to decrease XIAP mRNA levels at 6-9 hours in therange of 400-1200 nM concentrations. Exemplary oligonucleotides areshown in Table 5. TABLE 5 SEQ ID Oligonucleotide Sequence* NO: F3 ASATCTTCTCTTGAAAATAGG (PS) 278 F3 AS AU CTTCTCTTGAAAATA GG (2 × PS/ 279PO) F3 RP GGATAAAAGTTCTCTTCTA (PS) 280 G4 AS GCTGAGTCTCCATATTGCC (PS)281 G4 AS GC TGAGTCTCCATATTG CC (2 × PS/ 282 PO) G4 SCGGCTCTTTGCCCACTGAAT (PS) 283 C5 AS ACCATTCTGGATACCAGAA (PS) 284 C5 AS ACCATTCTGGATACCAG AA (2 × PS/ 285 PO) C5 RP AAGACCATAGGTCTTACCA (PS) 286AB6 AS GGGTTCCTCGGGTATATGG (PS) 287 AB6 RP GGTATATGGCGTCCTTGGG (PS) 288DE4 AS GGTATCTCCTTCACCAGTA (PS) 289 DE4 RP ATGACCACTTCCTCTATGG (PS) 290D7 AS GATTCACTTCGAATATTAA (PS) 291 D7 RP AATTATAACGTTCACTTAG (PS) 292*Bold residues = DNA residues with phosphorothioate linkages, underlinedresidues = 2′-O-methyl RNA bases, plain type = phosphodiester DNAresidues.

Tumor Cell Line and Animal Xenografts Model

The human non-small cell lung cancer cell line (large cell type)NCI-H460 (H460) was obtained from ATCC and maintained in RPMI 1640supplemented with 10% FCS at 37° C. in a humidified atmospherecontaining 5% CO₂. Cells were used in exponential growth phase, up to amaximum of 25 in vitro passages. Male SCID-RAG2 mice (7-9 weeks old,23-26g) were obtained from British Columbia Cancer Agency Joint AnimalFacility breeding colony and kept in aseptic environments. A tumor modelof NCI-H460 cells in SCID-RAG2 mice was established by subcutaneousimplantation of 1×10⁶ NCI-H460 cells on the back of mice.

Treatment of Cells With Antisense and Anticancer Drugs

One day prior to transfection, H460 cells were plated in 6- or 96-welltissue culture plates. Phosphorothioate antisense oligonucleotides weredelivered into cells with Lipofectamine 2000 (Life Technologies) in theform of liposome-oligonucleotide complexes. Following a 4.5 or 6 htransfection, the transfection medium was replaced with RPMI mediumcontaining 10% FBS, and the cells incubated for another 24 or 48 h.

Real-Time Quantitative RT-PCR

Total RNA from H460 cells treated with liposome-oligonucleotidecomplexes for 6 hours was immediately isolated using RNeasy mini spincolumns and DNase treatment (QIAGEN, Valencia, Calif.). Specific XIAPmRNA was measured using a real-time quantitative RT-PCR method. XIAPforward and reverse primers (600 nM) and probe (200 nM)(5′-GGTGATAAAGTAAAGTGCTTTCACTGT-3′ (SEQ ID NO 293), 6FAM-CAACATGCTAAATGGTTCCAGGGTGCAAATATC-TAMRA (SEQ ID NO: 294), and5′-TCAGTAGTTCTTACCAGACACTCCTCAA-3′ (SEQ ID NO: 295) were designed tospan exon 3-4 and 4-5 junctions. One of the primers, as well as theprobe, was designed to overlap an intron-exon boundary to blockdetection of any possible genomic DNA contamination. The RNA wasreverse-transcribed and PCR amplified using the TaqMan EZ RT-PCR kit(PE/ABI, Foster City, Calif.) in the ABI prism 7700 Sequence DetectionSystem (PE/ABI). The thermal cycling condition for the RT step were 50°C. for 2 min, 60° C. for 30 min, and 95° C. for 5 min, followed by 45cycles of PCR (at 94° C. for 20 s and 60° C. for 1 min per cycle). TheXIAP mRNA level of each sample was calculated relative to untreatedcontrol cells. XIAP mRNA levels were determined by the cycle thresholdmethod (Ct) using a threshold of 30× the baseline SD, and XIAP levelswere normalized for GAPDH content, using PE/ABI supplied primers andprobe.

Western Blot Analysis

The cells or tumor tissue samples were lysed with ice-cold lysis buffer(50 mM Tris, 150 mM NaCl, 2.5 mM EDTA, 0.1% SDS, 0.5% sodiumdeoxycholate, 1% NP-40, 0.02% sodium azide) containing proteaseinhibitors (Complete-Mini protease inhibitor tablets; BoehringerMannheim GmBH, Mannheim, Germany). After incubation for 30 min on ice,samples were centrifuged at 10,000 rpm for 15 min, and stored at −20° C.Protein content in the lysed extracts was determined using adetergent-compatible Bio-Rad assay (Bio-Rad Labs, Hercules, Calif.).Equal amounts of protein (40 μg/lane) were separated on 12%SDS-polyacrylamide gels or 4-15% gradient SDS-polyacrylamide pre-madegels (Bio-Rad) and transferred to nitrocellulose membranes. Primaryantibodies against XIAP, Bcl-2 (DAKO, Glostrup, Denmark), Bax (Sigma,St. Louis, Mo.), β-actin (Sigma), caspase-3 (BD PharMingen, San Diego,Calif.), and PARP (BD PharMingen) were used. The secondary antibody wasthe appropriate horseradish-conjugated anti-mouse or anti-rabbit IgG(Promega, Madison, Wis.). Proteins were detected by enhancedchemiluminescence (ECL; Amersham Pharmacia Biotech, Buckinghamshire,England) and visualized after exposure to Kodak autoradiography film.Scanning densitometry (Molecular Dynamics, Sunnyvale, Calif.) wasperformed to quantify band intensities by volume/area integration. Theamount of XIAP, caspase-3, Bcl-2 and Bax in cells was normalized totheir respective lane β-actin levels, upon stripping and reprobing.

Measurement of Cell Growth and Viability or Death

Growth inhibition of H640 cells was determined by the colorimetric MTTcell viability/proliferation assay. In brief, cells were treated withliposome-oligonucleotide complexes for 4.5 h, then incubated for another48 h at 37° C. in medium without transfection reagent oroligonucleotides in the presence or absence of anticancer drugs. MTT (25μg/well) was added to each well, and the plates incubated for 3 h at 37°C. Following the incubation step, the colored formazan product wasdissolved by the addition of 200 μl DMSO. Plates were read using themicrotiter plate reader (Dynex Technologies Inc., Chantilly, Va.) at awavelength of 570 nm. The percentage of surviving cells in wells treatedwith oligonucleotides was normalized to untreated controls. All assayswere performed in triplicate.

Apoptotic Flow Cytometric Assays

Cells were treated with liposome-oligonucleotide complexes for 4.5 h,and incubated for another 48 h in the medium without transfectionreagent at 37° C. Following incubation, cells were harvested, washedtwice with sample buffer (0.5% glucose in PBS without Ca⁺⁺ and Mg⁺⁺),and fixed in cold 70% ethanol at 4° C. for at least 18 hrs. Samples werecentrifuged at 3000 rpm for 10 min, then resuspended in sample buffercontaining 50 μg/ml propidium iodide (PI) and 400 U/ml RNase A. Sampleswere incubated for 30 min at room temperature and 30 min on ice,followed by flow cytometry analysis. EXPO Software (Applied CytometrySystems, Sacramento, Calif.) was used to generate histograms, which wereused to determine the cell cycle phase distribution after debrisexclusion. The Sub G1/G0 cell fraction was considered as representativefor apoptotic cells.

Nuclear Morphology

Cells were treated with liposome-oligonucleotde complexes for 4.5 h, andincubated for another 48 h at 37° C. in the medium without transfectionreagent or oligonucleotides. Cells were harvested and stained with 0.10μg/ml DAPI (4′,6-diamidino-2-2-phenylindole) for 30 min at roomtemperature. Cells were placed on a glass slide, cytospun, and viewedwith a Leica microscope and 40× objective lens under UV fluorescentillumination. Digital images were captured using Imagedatabase V. 4.01Software (Leica, Germany).

In Vivo Antitumor Activity

Efficacy experiments were conducted in male RAG2 immunodeficient micebearing NCI-H460 tumours or female RAG2 mice bearing LCC6 tumors.Treatments were commenced on day 3 after tumor inoculation. Saline(controls), G4 AS oligonucleotides (5 or 15 mg/kg), or G4 SColigonucleotides (5 or 15 mg/kg) were administered i.p. daily for fivedoses a week over a three week regimen. Vinorelbine (VNB, 5 or 10 mg/kg)was administered i.v. via the tail vein, either alone or in combinationwith oligonucleotides, at day 3, 7, 11 and 17 after tumor inoculation.When oligonucleotides were administered in combination with VNB, thedrug.treat.ment was performed four hours after ODN treatment.

Mice were observed daily. Body weight measurements and signs of stress(e.g., lethargy, ruffled coat, ataxia) were used to detect possibletoxicities. Animals with ulcerated tumor, or tumor volumes of 1 cm³ orgreater were killed. Digital caliper measurements of tumors wereconverted into mean tumor size (cm³) using the formula: ½[length(cm)]×[width (cm)]². An average tumor size per mouse was used tocalculate the group mean tumor size±SE (n=6 mice) from at least twoindependent experiments per group.

Tumor and Tissue Processing

Mouse tumors were collected on day 21 or 24 post-tumor inoculation andtreatment. One portion of the tumor tissue was fixed in formalin.Paraffin-embedded tissues were sectioned and subjected to grosshistopathology using hematoxylin and eosin staining andimmunohistochemistry for ubiquitin expression. The other portion of thetumor was homogenized in lysis buffer for western blot analysis (seeabove).

Statistical Analyses

Student's t test was used to measure statistical significance betweentwo treatment groups. Multiple comparisons were done using one-way ANOVAand a post-hoc test that compared different treatment groups by theShelle test criteria (Statistica release 4.5, StatSoft Inc., Tulsa,Okla.). Data were considered significant for a P-value of <0.05.

EXAMPLE 14 Antisense HIAP1 Oligonucleotides Decrease HIAP1 RNA andPolypeptide Expression

A library of 15 HIAP1 antisense nucleobase oligomers as oligonucleotideswas screened for protein knock-down by western blot analysis and for RNAknock-down by TaqMan, using the primers and probes described in Example3, above, under two different conditions. HIAP1 RNA levels may bedetected using standard Northern blot analyses or RT-PCR techniques. Theantisense oligonucleotides were administered to cells under basalconditions or under cycloheximide-induction conditions (24 hourtreatment with sub-toxic doses). Cycloheximide (CHX) can lead to a 10-to 200-fold induction of HIAP1 mRNA depending on the cell line treated.This in turn leads to an increase in HIAP1 protein, as seen on a Westernblot (70 kDa band). This effect of CHX is via two distinct mechanisms ofaction. First, CHX activates NFkB, a known transcriptional inducer ofHIAP1, by blocking the de novo synthesis of a labile protein, IkB, whichis an inhibitor of NFKB. This effect is mimicked by puromycin, anotherprotein synthesis inhibitor, and by TNF-alpha, which induces a signalingcascade leading to the phosphorylation, ubiquination, and degradation ofIkB. Only CHX leads to a further stabilization of the HIAP1 mRNA, asseen by the decreased rate of disappearance of HIAP1 message in thepresence of actinomycin D, to block de novo transcription, and CHX, asopposed to actinomycin D and puromycin or TNF-alpha combined.

SF295 glioblastoma cells were transfected with lipofectin andoligonucleotide (scrambled survivin, no oligonucleotide, antisense APO 1to APO 15) or left untreated. RNA was isolated from the cells six hoursafter transfection and the level of HIAP1 mRNA was measured byquantitative PCR (TaqMan analysis), normalized for GAPDH mRNA, with thevalue for the scrambled survivin oligonucleotide transfection set as1.0. The results of this experiment, a compilation of three separateexperiments, are shown in FIG. 21. The scrambled survivinoligonucleotide, the mock transfection, and untreated (non-transfected)cells, all showed similar HIAP1 1mRNA levels. Of the 15 antisenseoligonucleotides, seven (APO 1, 2, 7, 8, 9, 12, 15) showed an almost 50%decrease when compared to mock transfection or survivin scrambledcontrol oligonucleotide transfection (5′-mUmAmAGCTGTTCTATGTGmUmUmC-3′;SEQ ID NO: 296) (FIG. 21). Some of the oligonucleotides led to aninduction in HIAP1 mRNA, which may be a stress response to anon-specific toxic oligonucleotide. An antisense oligonucleotide maystill be effective at knocking down HIAP1 protein levels even if themessage is increased if the oligonucleotide is able to interfere withthe translation process.

The effect of HIAP1 antisense nucleobase oligomers on HIAP1 protein andmRNA expression was also examined in cells induced to express HIAP1.SF295 cells were transfected with oligonucleotides, or were mocktransfected. The transfected cells were then treated with 10 μg/mlcycloheximide for 24 hours to induce 70 kDa HIAP1 mRNA and protein.Protein levels were measured by western blot analysis with an anti-HIAP1polyclonal antibody, and normalized against actin protein in are-probing of the same blots. Scans of the western blot results areshown in FIG. 22A. The densitometric scan results were plotted againstthe mock results (set at 100%) in FIG. 22B. A line is drawn at 50% toeasily identify the most effective antisense oligonucleotides. Thetransfection process itself (e.g., mock or scrambled survivin) inducesHIAP1 protein compared to the untreated sample as shown on the westernimmunoblot.

Of the 15 tested nucleobase oligomers, six of them (APO 1, 2, 7, 8, 12,and 15) showed high activity, or had significant activity in both theprotein and mRNA assays, and did not cause a stress-induced increase inHIAP1 mRNA, such as that seen with APO 4, 6, 11, 13, 14 (FIG. 21), andby control oligonucleotides to APO 2 (mismatch or reverse polarity, seetext below and FIGS. 23 and 24). Note that APO 6 also showed evidence oftoxicity as seen by the general decrease in total protein (FIG. 23).

To further investigate the efficacy of HIAP1 antisense oligonucleotidesunder cycloheximide induction conditions, changes in HIAP1 mRNA weremeasured by TaqMan real time PCR 6 hours after transfection with APO 2,which targets an Alu repeat within an intron of HIAP1 and results in thegreatest block of CHX-induced upregulation of HIAP1 mRNA and protein.Controls for this experiment were three oligonucleotides for APO 2: onescrambled sequence (same base composition but random order,5′-AAGGGCGGCGGAGTGAGAC-3′; SEQ ID NO: 297), one reverse polarity (samebase composition, same sequential order but in the opposite direction,5′-AGAGG ACGGAGTCGGAGGC-3′; SEQ ID NO: 298), and one mismatch sequence(containing four base mismatches out of 19 bases,5′-CGGAGCGTGAGGATGGAGA-3′; SEQ ID NO: 299).

Transfection of the APO 2 antisense into cells resulted in a 50%decrease in mRNA compared to a scrambled survivin control and matchedperfectly with the protein results, while the scrambled control for APO2 (H1 sc apo 2 in FIG. 24) did not change HIAP1 mRNA levels at all(repeated twice here, and in two different experiments). However, themismatch control ODN (H1 mm apo 2) and the reverse polarity controloligonucleotide (H1 RV apo 2) showed an induction of 6 to 7 fold inHIAP1 mRNA at 6 hours. These oligonucleotides no longer targeted HIAP1,as expected, but may still target Alu repeats because of the degeneracyand repeat nature of these sequences. Therefore, it is possible thatthese two controls are toxic to the cell and cause a stress responsethat leads to the induction of HIAP1. This effect may also occur withthe antisense APO 2 oligonucleotide, but in this case, APO 2 also causesthe degradation of the induced HIAP1 mRNA which results in a relativedecrease of HIAP1 mRNA, compared to a scrambled survivin control, aswell as decreasing the relative fold induction of HIAP1 protein aftertransfection and CHX treatment, compared to scrambled survivin controloligonucleotide.

The six antisense HIAP1 nucleobase oligomers include two very effectiveoligonucleotides against an intronic sequence (APO 1, and APO 2, withAPO 2 demonstrating the better activity). These oligonucleotides couldbe used therapeutically for treatment of cancer or autoimmune disorders.The oligonucleotides against an intronic sequence would likely onlytarget pre-mRNA (very short-lived target) and not the mature, processedform of HIAP1. Typically, introns are not targeted for antisense exceptwhen one wants to alter splicing by targeting the intron-exon boundariesor the branching point. These usually result in the skipping of an exonrather than RNase-mediated degradation of the message. Both mechanismswould likely be favorable for the enhancement of apoptosis, as theskipping would result in the loss of the exon encoding the first twoimportant BIR domains of HIAP1. The APO 2 antisense ODN also targets anintron of survivin for 18 consecutive bases out of 19, but we did notsee any loss of survivin protein; only HIAP1 was decreased after theoligo treatment, demonstrating the specificity of the HIAP1 antisenseoligonucleotide. These antisense oligonucleotides hit Alu sequences inthe HIAP1 intron and potentially in many other genes, and induce thecancer cells to die (see below), which may be as a result of downregulating HIAP1 and some other critical genes, and thus of therapeuticvalue if it is not too toxic to normal cells.

Cancer cells have reportedly more Alu-containing transcripts and maytherefore be more sensitive to apoptosis induction with an Alu targetingnucleobase oligomer. Furthermore, this killing effect of nucleobaseoligomers APO 1 and APO 2 may be due to the combined effect of bothtargeting Alu sequences and HIAP1 simultaneously. This dual effect wouldresult in an effective way to prevent the normal stress response ofHIAP1 induction through the NFKB pathway, when the cell is exposed tocertain toxic agents. This stress response is most likely part of thecancer cell's anti-apoptotic program. By blocking HIAP1 expression, wecounter this anti-apoptotic stress response and precipitate the cancercell's demise.

EXAMPLE 15 HIAP1 Antisense Oligonucleotides Increase Cytotoxicity andChemosensitization

The effect of HIAP1 antisense nucleobase oligomers on thechemosentization of SF295 cells was also evaluated. Cells weretransfected with one of three different antisense oligonucleotides (APO7, APO 15, and SC APO 2 (control)). Twenty-four hours after transfectionwith the oligonucleotides, the cells were incubated with adriamycin foran additional 24 hours before assaying by for cell survival by assayingWST-1.

The WST-1 survival curves for SF295 cells transfected with theabove-described HIAP1 oligonucleotides and then treated with increasingconcentrations of adriamycin are shown in FIG. 25. The twooligonucleotides that resulted in a decrease in HIAP1 mRNA also showed adecrease in survival when treated with adriamycin compared to cellstreated with an oligonucleotide that did not reduce HIAP1 mRNA levels.Therefore, reducing HIAP1 levels by antisense, or other means, canchemosensitize a glioblastoma cell line that is highly resistant to thecytotoxic action of many chemotherapeutic drugs.

An additional 89 HIAP1 antisense sequences that can be employed in themethods of the invention are shown in Table 6. Sequences that are 100%identical between human HIAP1 and human HIAP2, or have one or twomismatches, are in bold. TABLE 6 Nucleobase oligomer sequence SEQ ID NO:AGCAAGGACAAGCCCAGTC 300 TGTAAACCTGCTGCCCAGA 301 AGAAGTCGTTTTCCTCCTT 302CCGAGATTAGACTAAGTCC 303 ACTTTTCCTTTATTTCCAC 304 TCCCAAACACAGGTACTAT 305CATTCTCAGCGGTAACAGC 306 ACCATCATTCTCATCCTCA 307 AATGTAACCTTCAACCATC 308TTTGTATTCATCACTGTC 309 TCACATCTCATTACCAAC 310 CCAGGTGGCAGGAGAAACA 311TGCAGACTTCAATGCTTTG 312 TAAGCAAGTCACTGTGGCT 313 CTGAGTCGATAATACTAGC 314ACTAGCCATTAGTAAAGAG 315 CAACAGCAGAGACCTTGTC 316 ATAGCATACCTTGAACCAG 317CATCTGTAGGCTAAGATGG 318 AGTTACCAGATGCCATCTG 319 AATCTACTCTGATAGTGGA 320GTTTCTGAAGCCAACATCA 321 TCAACTTATCACCTCCTGA 322 AAGAACTAACATTGTAGAG 323GTAGACAACAGGTGCTGCA 324 ATGTCCTCTGTAATTATGG 325 TACTTGGCTAGAACATGGA 326GAAGCAACTCAATGTTAAG 327 TTTGGTCTTTTGGACTCAG 328 CCATAGATCATCAGGAATA 329CAGGACTGGCTAACACATC 330 TTTAATGGCAGGCATCTCC 331 TTAAGCCATCAGGATGCCA 332GCTACAGAGTAAGCTGTGT 333 CTCTAGGGAGGTAGTTTTG 334 AAGAAAAGGGACTAGCCTT 335CAGTTCACATGACAAGTCG 336 GACTCCTTTCTGAGACAGG 337 ATTCACACCAGTGTAATAG 338CAGAAGCATTTGACCTTGT 339 CCAGCATCAGGCCACAACA 340 TTTCAGTAGGACTGTCTCC 341TGCAGCTAGGATACAACTT 342 AGAGGTAGCTTCCAAGTTG 343 GAAGTAATGAGTGTGTGGA 344GGATTTGATGGAGAGTTTG 345 GAACTTCTCATCAAGGCAG 346 AGGTCCTATGTAGTAAAAG 347CAATTTTCCACCACAGGCA 348 CATTATCCTTCGGTTCCCA 349 CTCAGGTGTTCTGACATAG 350GCTCAGATTAGAAACTGTG 351 CTGCATGTGTCTGCATGCT 352 TTAACTAGAACACTAGAGG 353CATAATAAAAACCCGCACT 354 CACCATCACAGCAAAAGCA 355 CTCCAGATTCCCAACACCT 356GGAAACCACTTGGCATGTT 357 GTTCAAGTAGATGAGGGTA 358 GATAATTGATGACTCTGCA 359ATGGTCTTCTCCAGGTTCA 360 GCATTAATCACAGGGGTAT 361 TAAAGCCCATTTCCACGGC 362TGTTTTACCAGGCTTCTAC 363 GATTTTTCTCTGAACTGTC 364 CTATAATTCTCTCCAGTTG 365ACACAAGATCATTGACTAG 366 TCTGCATTGAGTAAGTCTA 367 TCTTTTTCCTCAGTTGCTC 368GTGCCATTCTATTCTTCCG 369 GTAGACTATCCAGGATTGG 370 AGTTCTCTTGCTTGTAAAG 371TCGTATCAATCAGTTCTCT 372 GCAGAGAGTTTCTGAATAC 373 ATGTCCTGTTGCACAAATA 374CTGAAACATCTTCTGTGGG 375 TTTCTTCTTGTAGTCTCCG 376 CTTCTTTGTCCATACACAC 377GGAATAAACACTATGGACA 378 CATACTACTAGATGACCAC 379 TGTACCCTTGATTGTACTC 380GAAATGTACGAACTGTACC 381 GATGTTTTGGTTCTTCTTC 382 CTATCATTCTCTTAGTTTC 383ACACCTGGCTTCATGTTCC 384 GACTACAGGCACATACCAC 385 TGCCTCAGCCTGGGACTAC 386AGGATGGATTCAAACTCCT 387 GAGAAATGTGTCCCTGGTG 388 GCCACAACAGAAGCATTTG 389

We also analyzed human HIAP2 for sequences suitable for use as antisensenucleobase oligomers. Identified sequences are shown in Table 7. TABLE 7Nucleobase oligomer sequence SEQ ID NO: TTCTGAAAACTCTTCAATG 390CTTAGCATAAAGTATCAGT 391 CAAAAAAGTACTGCTTAGC 392 CAAGATAAAACTTGTCCTT 393TATCAGTCATGTTGTAAAC 394 CTAAATAACCTGTTCATCA 395 AGCACACTTTTTACACTGC 396ACCACTATTATTCTTGATC 397 TGTATTTGTTTCCATTTCC 398 ACTGTAAACTCTATCTTTG 399CTTAAGTGGGCTAAATTAC 400 CCTTCATATGGTCACACTA 401 GGTTACAAGCTATGAAGCC 402CTAAGCAACTATAGAATAC 403 TCCTTGATTTTTCACAGAG 404 ATACTAACTTAAAGCCCTG 405GGGTTGTAGTAACTCTTTC 406 TAGAACACAACTCTTTGGG 407 CTCTGAATTTCCAAGATAC 408TTTACTGGATTTATCTCAG 409 TGAGTAGGTGACAGTGCTG 410 GGAGGCAGTTTTGTGCATG 411CTATCTTCCATTATACTCT 412 TTGTTTGTTGCTGTTTGTC 413 TCCTTTCTGAGACAGGCAC 414ACCACCACGAGCAAGACTC 415 ACCTTGTCATTCACACCAG 416 TCCAGTTATCCAGCATCAG 417GCTTTTGAATAGGACTGTC 418 GAGATGTCTTCAACTGCTC 419 GGGGTTAGTCCTCGATGAA 420TCATTGCATAACTGTAGGG 421 GCTCTTGCCAATTCTGATG 422 ACCCTATCTCCAGGTCCTA 423ACAGGCAAAGCAGGCTACC 424 GTTCTGACATAGCATCATC 425 CTCAGAGTTTCTAGAGAAT 426ATGTTCTCATTCGAGCTGC 427 TGAACTGGAACACTAGATG 428 GCTCAGGCTGAACTGGAAC 429TTGACATCATCATTGCGAC 430 ACCATCACAACAAAAGCAT 431 CCACTTGGCATGTTCTACC 432TCGTATCAAGAACTCACAC 433 GGTATCTGAAGTTGACAAC 434 TTTCTTCTCCAGTGGTATC 435TTCTCCAGGTCCAAAATGA 436 ACAGCATCTTCTGAAGAAC 437 CACAGGTGTATTCATCATG 438CCAGGTCTCTATTAAAGCC 439 TTCTCTCCAGTTGTCAGGA 440 GAAGTGCTGACACAATATC 441TTTTCCTTCTCCTCCTCTC 442 CATCTGATGCCATTTCTTC 443 AGCCATTCTGTTCTTCCGA 444CCAGGATAGGAAGCACACA 445 ATGGTATCAATCAGTTCTC 446 CCGCAGCATTTCCTTTAAC 447CAGTTTTTGAAGATGTTGG 448 GTGACAGACCTGAAACATC 449 GGGCATTTTCTTAGAGAAG 450AGTACCCTTGATTATACCC 451 GAAATGTACGAACAGTACC 452 TGAAAAACTCATAATTCCC 453CCATCTTTTCAGAAACAAG 454 CTATAATTCTCTCCAGTTG 455 CTCCCTTAGGTACACATAC 456ACAAGCAGTGACACTACTC 457 GTAACTCCTGAAATGATGC 458 CAACAAATCCAGTAACTCC 459CACCATAACTCTGATGAAC 460

Other Embodiments

All publications and patent applications mentioned in thisspecification, including U.S. Pat. Nos. 5,919,912, 6,156,535, and6,133,437, are herein incorporated by reference to the same extent as ifeach independent publication or patent application was specifically andindividually indicated to be incorporated by reference.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure come within known or customary practice within theart to which the invention pertains and may be applied to the essentialfeatures hereinbefore set forth.

1. A method of treating an animal having a lymphoproliferative disorder, the method comprising: administering to the animal in need thereof an IAP nucleobase oligomer of up to 30 nucleobases in length, the nucleobase oligomer comprising at least eight consecutive nucleobases of SEQ ID NOs: 1-96, 97-162 and 278-292, thereby treating the animal.
 2. The method, according to claim 1, in which the nucleobase oligomer comprises at least eight consecutive nucleobases of a sequence selected from the group consisting of SEQ ID NOs: 1-96, 97-162 and 278-292.
 3. The method, according to claim 2, in which the nucleobase oligomer comprises at least eight consecutive nucleobases of a sequence selected from the group consisting of SEQ ID NOs: 1-96, 97-162 and 278-292.
 4. The method, according to claim 3, in which the nucleobase oligomer consists essentially of a sequence selected from the group consisting of SEQ ID NOs: 1-96, 97-162 and 278-292.
 5. The method, according to claim 4, in which the nucleobase oligomer consists of a sequence selected from the group consisting of SEQ ID NOs: 1-96, 97-162 and 278-292.
 6. The method, according to claim 1, in which the nucleobase oligomer is an oligonucleotide.
 7. The method, according to claim 6, in which the oligonucleotide comprises at least one modified linkage.
 8. The method, according to claim 7, in which the modified linkage is selected from the group consisting of phosphorothioate, methylphosphonate, phosphotriester, phosphorodithioate, and phosphoselenate linkages.
 9. The method, according to claim 1, in which the nucleobase oligomer comprises at least one modified sugar moiety.
 10. The method, according to claim 9, in which the modified sugar moiety is a 2′-O-methyl group or a 2′-O-methoxyethyl group.
 11. The method, according to claim 1, in which the nucleobase oligomer comprises at least one modified nucleobase.
 12. The method, according to claim 11, in which the modified nucleobase is 5-methyl cytosine.
 13. The method, according to claim 1, in which the nucleobase oligomer is a chimeric nucleobase oligomer.
 14. The method, according to claim 13, in which the nucleobase oligomer comprises DNA residues linked together by phosphorothioate linkages, the DNA residues flanked on each side by at least one 2′-O-methyl or 2′-O-methoxyethyl RNA residue.
 15. The method, according to claim 14, in which the DNA residues are flanked on each side by at least three 2′-O-methyl or 2′-O-methoxyethyl RNA residues.
 16. The method, according to claim 15, in which the DNA residues are flanked on each side by four 2′-O-methyl or 2′-O-methoxyethyl RNA residues.
 17. The method, according to claim 14, in which the RNA residues are linked together by phosphorothioate linkages, and the RNA residues are linked to the DNA residues by phosphorothioate linkages.
 18. The method, according to claim 13, in which the nucleobase oligomer comprises DNA residues linked together by phosphodiester linkages, the DNA residues flanked on each side by at least two 2′-O-methyl or 2′-O-methoxyethyl RNA residues linked together by phosphorothioate linkages.
 19. The method, according to claim 18, in which the DNA residues are flanked on each side by at least three 2′-O-methyl or 2′-O-methoxyethyl RNA residues.
 20. The nucleobase oligomer of claim 1, the nucleobase oligomer comprising eleven DNA residues flanked on each side by four 2′-O-methyl RNA residues, the nucleobase oligomer consisting of SEQ ID NOs: 16, 27, 41, 47, 51,63,141, 151,155, 157 and 161, the residues linked together by phosphorothioate linkages.
 21. The method of claim 1, further comprising administering to the animal a biological response-modifying agent.
 22. The method, according to claim 21, in which the biological response-modifying agent is an interferon.
 23. The method, according to claim 22, in which the interferon is interferon alpha, interferon beta, or interferon gamma.
 24. The method, according to claim 1, in which the lymphoproliferative disorder is multiple sclerosis, Crohn's disease, lupus erythematosis, rheumatoid arthritis, or osteoarthritis.
 25. The method, according to claim 24, in which the lymphoproliferative disorder is multiple sclerosis.
 26. The method, according to claim 1, in which the nucleobase oligomer is administered to the animal intravenously.
 27. The method, according to claim 1, in which the animal is a human.
 28. A composition comprising: (i) an IAP nucleobase oligomer of up to 30 nucleobases in length, the nucleobase oligomer comprising at least eight consecutive nucleobases of SEQ ID NOs: 1-96, 97-162 and 278-292; and (ii) a biological response-modifying agent, in amounts that together are sufficient to treat an animal having a lymphoproliferative disorder.
 29. The composition, according to claim 28, in which the nucleobase oligomer comprises at least eight consecutive nucleobases of a sequence selected from the group consisting of: SEQ ID NOs: 1-96, 97-162 and 278-292.
 30. The composition, according to claim 29, in which the nucleobase oligomer comprises at least eight consecutive nucleobases of a sequence selected from the group consisting of SEQ ID NOs: 1-96, 97-162 and 278-292.
 31. The composition, according to claim 30, in which the nucleobase oligomer consists essentially of a sequence selected from the group consisting of SEQ ID NOs: 1-96, 97-162 and 278-292.
 32. The composition of claim 31, in which the nucleobase oligomer consists of a sequence selected from the group consisting of SEQ ID NOs: 1-96, 97-162 and 278-292.
 33. A method of enhancing apoptosis of a cell in an animal, the method comprising: administering to the animal the composition, according to claim 28, in amounts that inhibits expression of an IAP in the cell.
 34. The method, according to claim 33, in which the cell is in vivo.
 35. The method, according to claim 33, in which the cell is ex vivo.
 36. The method, according to claim 33, in which the cell is of lymphoid origin.
 37. The method, according to claim 33, in which the cell is a T cell.
 38. The method, according to claim 33, in which the cell is a B-cell.
 39. A method of treating a human having multiple sclerosis, the method comprising: administering to the patient in need thereof an IAP nucleobase oligomer of up to 30 nucleobases in length, the nucleobase oligomer comprising at least eight consecutive nucleobases of SEQ ID NOs: 1-96, 97-162 and 278-292, thereby treating the human.
 40. A method of treating a human having multiple sclerosis, the method comprising administering to the patient: (i) an IAP nucleobase oligomer of up to 30 nucleobases in length, the nucleobase oligomer comprising at least eight consecutive nucleobases of SEQ ID NOs: 1-96, 97-162 and 278-292; and (ii) an interferon in amounts that together are sufficient to treat the human.
 41. A method of treating an animal having a lymphoproliferative disorder, the method comprising administering to the animal an effective amount of a catalytic RNA molecule capable of cleaving XIAP mRNA, thereby treating the animal.
 42. The method, according to claim 41, in which the binding arms of the catalytic RNA molecule contain at least eight consecutive nucleobases corresponding to a sequence of any one of Tables 1 or
 2. 43. The method, according to claim 42, in which the RNA molecule is in a hammerhead motif.
 44. The method, according to claim 41, in which the RNA molecule is in a hairpin, hepatitis delta virus, group 1 intron, VS RNA or RNAseP RNA motif.
 45. A method of treating an animal having a lymphoproliferative disorder, the method comprising administering to the animal an effective amount of a double-stranded RNA molecule consisting of between 21 and 29 nucleobases, the RNA molecule comprising at least eight consecutive nucleobases corresponding to a sequence of any one of Tables 1 and
 2. 46. A method of treating an animal having a lymphoproliferative disorder, the method comprising administering to the animal an effective amount of a double-stranded hairpin RNA molecule consisting of between 50 and 70 nucleobases, the RNA molecule comprising a first domain of between 21 and nucleobases that comprise least eight consecutive nucleobases corresponding to a sequence of any one of Tables 1 and 2; a second domain complementary to the first domain, and a loop domain situated between the first and the second domains such that the first domain and the second domain are capable of duplexing to form the double-stranded hairpin RNA molecule. 